Characterization of clinical and immunological features in patients coinfected with dengue virus and HIV

    Characterization of clinical and immunological features in patients coinfected with dengue virus and HIV Amanda Torrentes-Carvalho, Eugˆenio Damaceno Hottz, Cintia Ferreira Marinho, J´essica Badolato-Corrˆea da Silva, Luzia Maria de Oliveira Pinto, Luciana Gomes Fialho, Fernando Augusto Bozza, Rivaldo Venˆancio Cunha, Paulo Vieira Damasco, Claire Fernandes Kubelka, Elzinandes Leal de Azeredo PII: DOI: Reference:

S1521-6616(16)30005-5 doi: 10.1016/j.clim.2016.01.005 YCLIM 7600

To appear in:

Clinical Immunology

Received date: Revised date: Accepted date:

14 September 2015 2 January 2016 14 January 2016

Please cite this article as: Amanda Torrentes-Carvalho, Eugˆenio Damaceno Hottz, Cintia Ferreira Marinho, J´essica Badolato-Corrˆea da Silva, Luzia Maria de Oliveira Pinto, Luciana Gomes Fialho, Fernando Augusto Bozza, Rivaldo Venˆ ancio Cunha, Paulo Vieira Damasco, Claire Fernandes Kubelka, Elzinandes Leal de Azeredo, Characterization of clinical and immunological features in patients coinfected with dengue virus and HIV, Clinical Immunology (2016), doi: 10.1016/j.clim.2016.01.005

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ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Characterization of clinical and immunological features in patients coinfected with Dengue virus and HIV

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Running Title: Immunological parameters in dengue and HIV coinfection

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Amanda Torrentes-Carvalhoa,*; Eugênio Damaceno Hottzb; Cintia Ferreira Marinhoa; Jéssica Badolato-Corrêa da Silvaa; Luzia Maria de Oliveira Pintoa; Luciana Gomes Fialhoa;Fernando

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Augusto Bozzab; Rivaldo Venâncio Cunhac; Paulo Vieira Damascod; Claire Fernandes Kubelkaa; Elzinandes Leal de Azeredoa

Laboratório de Imunologia Viral, Instituto Oswaldo Cruz / FIOCRUZ, Rio de Janeiro, Brazil

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Instituto de Pesquisa clínica Evandro Chagas (IPEC), Rio de Janeiro, Brazil

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Departamento de Clínica Médica, FM, Universidade Federal do Mato Grosso do Sul, Campo

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Grande, MS; Brazil

Hospital Universitário Gaffrée Guinle, Universidade Federal do Estado do Rio de Janeiro - UNI

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RIO, Rio de Janeiro, Brazil

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*Corresponding author

Av. Brasil, 4365 Manguinhos, Laboratório de Imunologia Viral, Pavilhão Hélio e Peggy Pereira,

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Rio de Janeiro, RJ, Brazil, CEP 2104-360. E-mail address: [email protected]; [email protected]

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Keywords: coinfection; lymphocytes; cytokines; dengue; HIV.

Abbreviations: AIDS, Acquired Immunodeficiency Syndrome; Ag, antigen; ANOVA, analysis of variance; APC, antigen-presenting cell; APC-Cy7, Allophycocyanin conjugated cyanine dye; BSA, bovine serum albumin; CD, cluster of differenciation; Cy, cycrome; DENV, dengue virus; DF, dengue fever; DHF/DSS, dengue haemorrhagic fever or shock syndrome; DMSO, dimethyl sulphoxide; ELISA, enzyme-linked immunosorbent assay; FACS, Fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HIV, human immunodeficiency virus; HLA, human leucocyte antigen; IFN, interferon; IL, interleukin; IP, induced protein; LPS, lipopolysaccharide; mAb, monoclonal antibody; MHC, major histocompatibility complex; NaN3, sodium azide; NF-κB, nuclear factor κB; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PE, phycoerythrin; PE-Cy7, phycoerythrin conjugated cyanine dye; RNA, ribonucleic acid; ROS, reactive oxygen species; TNF, tumor necrosis factor; RT-PCR, Reverse transcription polymerase chain reaction; WHO, World Health Organization. 1

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Summary The pathogenesis of dengue in subjects coinfected with HIV remains largely unknown. We investigate clinical and immunological parameters in coinfected DENV/HIV patients. According to

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the new dengue classification, most coinfected DENV/HIV patients presented mild clinical

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manifestations of dengue infection. Herein, we show that DENV/HIV coinfected patients had

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higher CD8 T cells percentages reflected as a lower CD4/CD8 ratio. Furthermore, CCR5 expression on CD4 T cells andCD107a expression on both T subsets were significantly higher in coinfected patients when compared with monoinfected DENV and HIV individuals respectively. Increased inflammatory response was observed in treated HAART coinfected patients despite undetectable

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HIV load. These data indicate that DENV infection may influence the clinical profile and immune

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response in individuals concomitantly infected with HIV.

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ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Introduction In tropical countries, numerous infectious diseases coexist, and therefore the possibilities of coinfections are common. In this context, infections with human immunodeficiency virus (1) and

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Dengue virus (DENV) are serious public health problems in tropical and subtropical regions and

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represent major public health problems in several countries, including Brazil. DENV belongs to the

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genus Flavivirus, family Flaviviridae, a small group of enveloped viruses (2) with a positive sense RNA virus that co-circulates in endemic regions as four closely-related distinct serotypes DENV-1, DENV-2, DENV-3 and DENV-4 (3). In various regions of Brazil, dengue epidemics occur for decades, continually contributing to the high rates of morbidity and mortality (4). In 2015 from

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January to July were reported 1.319.957cases of dengue in the countrywith1.092 severe cases and 600deaths (5).We have over 30 million people living with AIDS (Acquired Immune Deficiency

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Syndrome) caused by HIV. Since the 1980, it was reported 757000 cases of AIDS in Brazil, with detection ratio around 20.4 cases per 100 thousand inhabitants representing 39 000 new AIDS cases per year. In the last years there was an increase in the number of people who started antiretroviral

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treatment with 57000 in 2013 to 74000 new treatments in 2014 (6).

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HIV is a member of the genus Lentivirus, Retroviridae family and contains two singlestranded RNA copies of 9.2 kb comprising the HIV-1 and HIV-2 types. The natural history of HIV

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infection includes a long latency period, accompanied by the development of opportunist infections and deregulation of lymphocyte T CD4 cells. At the time when the CD4 frequency is very low, the patient goes into a severe immunodeficiency and opportunistic infections, which can lead to the

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death in the absence of treatment (7). Compared with HIV infection, the clinical course is quite different during dengue infection varying from an acute febrile illness usually benign to a severe form with hemodynamic disorders, severe hemorrhagic manifestations, cavity effusions and shock. Despite the growing knowledge about the dengue fever and its vector, the evolution of to severe clinical conditions is not fully understood. Several hypotheses are proposed to explain the pathogenesis of the disease. Among them, interacting factors: (i) environmental, uncontrolled growth of the population and higher incidence susceptible hosts to circulating serotypes; (ii) host, including secondary infections, genetics, nutritional status, age, comorbidities; and (iii) viral, such as viral genotype or serotype (8, 9). All of these factors are related to an increased viremia in the initial phase of dengue, the activation of innate and adaptive immunity leading to “cytokine storm” as well as to inflammatory mediators (10). Although, effectively different in relation to clinical evolution, DENV and HIV share some immunopathogenic mechanisms such as extensive activation of the immune system, cytokine storm and depleted blood T lymphocytes (9, 11, 12). Despite the vast knowledge about both diseases, only few coinfection cases have been reported around world so 3

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection far to enhance the understanding of the effects in the clinical presentation, immune response and severity. Most of them culminate in findings that DENV infection transiently reduces HIV viral loads, without no impact on the clinical course neither dengue nor AIDS (13-17). Nevertheless, no

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scientific report evaluated the immune response during DENV/HIV coinfection and the degree of

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interference between them. In this context, we evaluated the clinical and laboratorial parameters,

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cytokines as well as lymphocytes subsets during coinfection with DENV and HIV. We describe here that most DENV/HIV coinfected patients showed mild clinical manifestations of dengue. Most coinfected patients presented elevated CD8 T cells percentages and decreased CD4/CD8 ratios were observed in DENV/HIV coinfection as well as DENV

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monoinfection. In addition, increased cytokine/chemokine production was observed in both coinfected DENV/HIV and monoinfected DENV patients. Several questions are raised of the

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possible consequences for the patient by the simultaneous presence of two viral infections.

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Materials and Methods

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Study population and laboratorial diagnosis The study was carried out in Brazil from 2010 to 2011during DENV-1and 2outbreaks.

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Patients were assisted at Professora Hesterina Corsini-NHU Dia-Hospital Campo Grande/MS; Rio Laranjeiras Hospital/RJ; ward of the Infectious and Parasitic Diseases (DIP), ward of the Institute of Clinical Research Evandro Chagas (IPEC), Reference Center for Infectious and Parasitic Diseases

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(CEDIP) respectively. Blood samples from HIV positive individuals were collected at Universitary hospital Gaffrée Guinle, Universidade Federal do Estado do Rio de Janeiro - UNI RIO. During the study 95 cases of dengue were included and submitted to investigation. Participants were grouped according to HIV and DENV infection as follows: Seventy-four DENV monoinfected patients, 21 DENV/HIV coinfected patients and 14 HIV monoinfected patients. All infected HIV patients were receiving highly active antiretroviral therapy (HAART), according to the Brazilian guidelines. Ten individuals were enrolled in the study as healthy donors (HD) considering they had no episodes of fever for more than three months or a history of other diseases. Informed consent was obtained from all patients. Clinical classification of dengue was performed according to the World Health Organization/Brazilian Health Ministry WHO/ TDR– 2009 (WHO/TDR, 2009) criteria in the groups: dengue without warning signs (DwoWS), dengue with warning signals (DwWS) and severe dengue (SD). Patients with dengue were classified in accordance with or without the presence of warning signs assessed as clinical parameters such as abdominal pain or tenderness, persistent vomiting, 4

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection clinical fluid accumulation, mucosal bleed, lethargy, restlessness, liver enlargement associated to laboratory parameters as increase in haematocrit concurrent with rapid decrease in platelet count. Mild hemorrhagic manifestations like petechias and mucosal membrane bleeding (e.g. nose and

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gums). Patients considered as severe dengue fever presented severe clinical manifestations such as

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haemoconcentration, hypotension (postural hypotension with a decrease in the systolic arterial

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pressure of 20 mmHg in the supine position or a systolic arterial pressure of < 90 mmHg), plasma leakage leading to dengue shock and/or fluid accumulation with respiratory distress, severe hemorrhages showed by massive vaginal bleeding (in women of childbearing age) or gastrointestinal bleeding and/or severe organ impairment (hepatic damage, renal impairment,

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cardiomyopathy, encephalopathy or encephalitis).

Laboratorial characteristics from all patients were obtained in the hospitals in which they

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were treated. Data were correlated to the days of disease onset until the moment when the patients were interviewed. All DENV monoinfected and coinfected DENV/HIV patients in this research recovered from the illness uneventfully and survived to hospital discharge.

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The dengue diagnosis was confirmed by enzyme-linked immunosorbent assay ELISA-IgM

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(Panbio, inc, USA); detection of NS1 protein by Platelia Dengue NS1 antigen kit (18) and molecular diagnosis by RT-PCR (19). The immune response to Dengue was considered as primary

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or secondary by IgG ELISA according to previously established criteria (Focus Diagnosis, Cypress, CA, USA)(20). The HIV diagnosis was confirmed by Real Time HIV-1 Qualitative amplification assay (Abbott Molecular). HIV monoinfected patients had a previous diagnosis ambulatory

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monitoring in DIP from HUGG and IPEC.

Ethics statement

Procedures performed during this work were approved by the Ethics Committees from Instituto de Pesquisas Clinicas Evandro Chagas, Fiocruz (CAAE 3723.0.000.009-08); and Plataforma Brasil, Fiocruz (CAAE 13318113.7.0000.5248). All participating subjects gave written informed consent.

Human peripheral blood mononuclear cells (PBMCs) purification and cryopreservation Venous blood samples from patients were collected in tubes containing 3.2 to 4% citrate (BD Biosciences). PBMCs were isolated and purified by density gradient centrifugation (FicollPaque; Amersham Biosciences Corp, Piscataway, NJ) and ressuspended in 1ml of freezing solution [Fetal bovine serum - FBS (Gibco invitrogen Carlsbad, CA) plus 10% dimethyl sulfoxide(DMSO; Sigma)]and cryopreserved in liquid nitrogen. The plasma was aliquoted and stored at -70° C. 5

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Monoclonal antibodies Four-color flow cytometry method were performed using the following monoclonal

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antibodies: anti-CD4-Allophycocyanin conjugated cyanine dye (APC Cy7) (BioLegend; clone

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OKT4; Catalog 317417); anti-CD8 phycoerythrin conjugated cyanine dye (PE Cy7) (E- Bioscience;

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clone RPAT8; catalog 25-0088); anti-CD107aAlexa Fluor® 647 (E- Bioscience; clone CBIOH4A3; catalog 51107970) and anti-CCR5- phycoerythrin (PE) (R&D Systems; clone CTC5; catalog FAB1802). A matching isotype control for each antibody was included in all experiments.

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Extracellular labeling for flow cytometry analysis

Aliquots containing about 106 cryopreserved PBMCs from patients and healthy donors were

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thawed in water bath at 37oC, centrifuged (350 g, 5 min) and washed once with 1mL PBS pH 7.4 with 2% FBS and 0,01% NaN3. Multiple labeling of PBMCs was performed for flow cytometry analysis. Cells were labeled extracellularly according to previously described methods (20) with

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slight modifications. For analysis, 10,000-50,000 events were acquired on a FACS CyAn (Dako)

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and analyses were performed with the FlowJo version 7.6.1 software (Tree Star Inc., San Diego,

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CA).

Quantification of circulating cytokines and chemokines by immunoassays Plasma samples were obtained from DENV monoinfected patients (n=74), DENV/HIV

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coinfected (n=25), HIV monoinfected patients (n=14) and from HD (n=10). Levels of IL-1RA, IL17A, IL-10,IL-4, IL-6, IFN-γ, IL-8/CXCL8, RANTES/CCL5, IP-10/CXCL10 and MIP-1β/CCL4 were determined by ELISA(Peprotech; Quantikine, R&D Systems) and Multiplex immunoassay kits (eBioscense BMS203INST; ProcartaPlex Multiplex immunoassay;eBioscenseEPX01010420901) in compliance with manufacturer’s directions. Standard curves were prepared by serial dilutions of the aliquot corresponding to cytokine pattern supplied by the manufacturer. The determination of the optical density of samples and standards was performed using Expert ® Plus spectrophotometer using a wavelength of 620nm.

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ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Statistical Analysis Data were analyzed as medians and interquartile ranges. For non-parametric data sets, Kruskal-Wallis test followed by Dunn’s multiple comparison test were applied. Fisher test was used

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to compare clinical manifestations associated with dengue severity within different patient groups.

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The correlations were estimated by Spearman regression analysis. The P-values < 0.05 were

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considered to be statistically significant. Statistical analysis was performed using GraphPad Prism software, version 6.0 (GraphPad Software, San Diego, CA).A sample size calculation using the program StateMate (GraphPad Inc.).

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Results

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Study population

We prospectively included 95 patients with positive diagnosis of dengue infection in the acute phase (2–10 days after disease onset) during DENV-1and DENV- 2 outbreaks. Of these, 74

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patients were DENV monoinfected and 21 were DENV/HIV coinfected. Fourteen HIV

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monoinfected individuals and 10HDwere included in the analyses. Demographical, clinical and laboratorial data from the different patient groups enrolled in this study are shown in Table 1.

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No differences in age were found among the studied groups. Most DENV positive patients were IgG positive indicating a previous dengue infection (93%). All HIV infected patients (monoinfected and DENV coinfected) were receiving antiretroviral therapy (supplemental table), according to Brazilian guidelines, resulting in CD4

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counts above 200 cells/mm3 and viral loads< 50 copies, indicating a good response to treatment. Patients enrolled in the study presented fever equal to or greater than 38° C, accompanied by one or more of the following symptoms: myalgia, arthralgia, leukopenia, rash, headache, lymphadenopathy, nausea, vomiting, positive tourniquet test, thrombocytopenia, hepatomegaly, hemorrhagic manifestations or effusions. Based on the study of 74 DENV monoinfected patients, 41 presented no Warning Signs, 17 had WS and 16 were Severe DF. On the other hand, among the 21 DENV/HIV coinfected patients, 7 presented WS and one was classified as Severe DF (Table1). Thrombocytopenia is one of the most common hematologic manifestations observed during acute phase of dengue infection (21) but is also characteristic of HIV individuals (22).In general both groups of patients, monoinfected and coinfected, presented lower platelets when compared with healthy donors and HIV monoinfected individuals (Table 1). As expected, lower platelets counts were found in Severe DENV monoinfected patients when compared with those with WS or healthy individuals (Table 1). Nevertheless, DENV monoinfected patients presented significantly 7

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection lower platelets counts as compared to DENV/HIV coinfected patients [median x103/mm3 (95% CI): 126 (75-144) and 164 (106-214), respectively]. In addition, both co and DENV monoinfected patients showed lower leukocytes counts as

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compared with HD and HIV monoinfected patients. With respect to biochemical parameters, the

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circulating AST and ALT levels were not significantly different among the groups of study (Table

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1), although, 68%DENV monoinfected patients and 94% DENV/HIV coinfected showed ALT levels above normal reference values. Similarly, 77% DENV monoinfected patients and 71% DENV/HIV coinfected patients presented AST levels above normal reference values. Considering manifestations associated with a risk of an unfavorable outcome, the DENV

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monoinfected patients presented more plasma leakage, hemorrhagic manifestations as well as severe hemorrhagic manifestations (e.g. gastrointestinal and mucosal bleeding), representing 14 out

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72. According to the new dengue classification, WHO 2009, the diagnosis of severe cases was less frequent in DENV/HIV coinfected patients, 1 out 23 (Table 1 and Table 2).

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T-cell subsets

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Persistent CD8 T cell expansion, low CD4/CD8 cell ratios are observed in treated HIV patients and associated morbidity and mortality (23). So, we sought to determine T cell frequencies

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in all groups studied. We did not observe differences in the CD4 absolute counts between DENV/HIV coinfected patients or HIV monoinfected individuals [Median (95% confidence interval, CI): 451 (165-833) and 517 (304-1005), respectively]. One out 16 (6.2%) coinfected

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patients showed CD4 absolute counts less than 200 cells/mm3. In addition, no difference was found in the CD4 absolutes counts in DENV monoinfected patients [Median (95% confidence interval, CI): 621 (228-837)].

No significant difference was observed in the CD4 T lymphocyte percentages between mono and coinfected patients (table 1). However, we observed a significantly lower frequency in CD4 T lymphocytes in DENV monoinfected, DENV/HIV coinfected and HIV monoinfected individuals as compared to HD [median (95% CI): HD 43 (40-53, n=10); DENV 34 (22-42, n=22); DENV/HIV 22 (12-33, n=9); HIV 33 (20-41, n=12)]. In addition, the CD4/CD8 ratio was significantly lower in these same groups, and also in DENV monoinfected patients [median (95% CI): HD 2.0 (1.6-3.3, n=10); DENV 1.7 (0.6-2.2, n=22); DENV/HIV 0.7 (0.4-0.8, n=9); HIV 1.0 (0.5-2.0, n=12)]. We did not observe differences in the CD8 absolute counts among DENV/HIV coinfected patients, HIV monoinfected individuals [Median (95% confidence interval, CI): 654 (208-1113) and (420-965), respectively] or DENV monoinfected [Median (95% confidence interval, CI): 339 (2091014)]. Interestingly, we observed a higher frequency in the CD8 T cell subset in the coinfected 8

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection group as compared to HD [median (95% CI): HD 22 (15-27, n=10); DENV/HIV 35 (25-39, n=9), P<0.003]. These data are corroborated by the fall in CD4/CD8 ratio observed in DENV/HIV coinfected and HIV monoinfected compared with HD (Table 1).According to disease severity, both

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DENV monoinfected patients with WS as well as Severe DF patients had lower CD4/CD8 ratios as

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compared with HD. Similarly, DENV/HIV coinfected patients with WS also showed lower

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CD4/CD8 ratio when compared with HD (Table 1). It has been demonstrated that CD4/CD8 ratio below 1.0 is associated with persistent immune dysfunction (23). We observed that 7 out 23 (30.4 %) DENV monoinfected presented CD4/CD8 ratio below 1 and 5 out 12 (41.6%), HIV monoinfected and most coinfected patients presented a CD4/CD8 ratio below 1 (8 out 9, 88.8%).

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Finally, we demonstrated an increase in the frequency of double CD4+CD8+T cells in the DENV/HIV coinfected patients [5.5 (3.0-8.3), n=9] as compared to DENV monoinfected [3.4 (1.8-

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4.5), n=22, P<0.05], HIV monoinfected [2.3 (1.9-3.8), n=12, P<0.01] and HD [1.8 (1.1-3.0), n=10, P<0.0006].

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DENV/HIV coinfection increases the expression of chemokine receptor CCR5/HIV co-

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receptor on T lymphocytes from patients during dengue infection The chemokine receptor CCR5 is a co-receptor for HIV, thus plays an important role in HIV

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infection and replication. Importantly, CCR5 is expressed on T cells driving the migration of effector and memory T cells into inflammatory tissues (24). As we observed a lower frequency in percentages of CD4 T cells in the DENV/HIV coinfected compared with HD, we sought to evaluate

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the frequency of CCR5 on T lymphocytes in all groups of study. Representative flow cytometry data illustrates the distribution of cells labeled for CD4 and CCR5 (Figures 1 A-D). Collectively, the median percentage of positive cells in the CD4 and CD8 subsets are shown as separately labeling CCR5 cells in HD and subjects with DENV monoinfection, DENV/HIV coinfection or HIV monoinfection. As presented in the Figure 1 E, coinfected patients had a significant increase in the CCR5 expression among CD4 cells when compared to HD (P<0.001). Interestingly, coinfected patients showed a more pronounced increase in the CCR5 expression among T CD4 cells compared with DENV monoinfected patients (P<0.05). Increased expression of CCR5 among CD8 T cells was observed in monoinfected DENV patients compared with HD (P<0.05) and monoinfected HIV group (P<0.01) (Figure 1 F). Flow cytometry analyses are difficult to perform if we consider that the cells must be in good conservation, so the tested samples were small, mainly in the group of coinfected DENV/HIV patients .We calculated the power of our comparisons and we found a good power of sample sizes tested for the different assays used and most of them were higher than 0.9. 9

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Predominance of T lymphocytes with a cytotoxic profile in patients with dengue despite HIV infection We observed a higher frequency of CD8 T cells in the peripheral blood of DENV/HIV

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coinfected patients; therefore we also assessed the cytotoxic potential by the CD107a extracellular

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expression. As shown in Figures 1G, we observed an increase in T CD4 lymphocytes with cytotoxic

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profile among DENV monoinfected (P<0.01) and coinfected (P<0.001) groups as compared to controls. Additionally, coinfected patients presented an increased expression of CD107a among CD4 T cells as compared to HIV monoinfected group (P<0.01). With respect to the T CD8 subset, we demonstrated that coinfected DENV/HIV patients showed increased CD107a expression as

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compared with HD (P<0.001) and HIV monoinfected (P<0.01) groups (Figure 1H). DENV monoinfected patients showed a slight increase in CD107a but no significant difference was found.

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Interestingly, both CD4 and CD8 T subsets displayed similar patterns of CD107a expression, highlighting its higher frequency in coinfected cases.

We searched for double expression of the cytotoxic CD107a molecule and CCR5,

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characterizing migratory cytotoxic cells. Interesting, we showed a more pronounced increase of

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CD107a expression on CCR5 expressing CD4 and CD8 T cells in all groups of patients, including HD (Figure 1-I-J). These data suggests a potential ability of T cells to exert effector function and

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migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment with cytotoxic function to inflammatory tissues during dengue.

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Evaluation of natural ligands for CCR5, MIP1β/CCL4 and RANTES/CCL5 in monoinfected DENV, coinfected DENV/HIV and monoinfected HIV RANTES/CCL5 (regulated on activation, normal T-cell expressed and secreted) and MIP1β/CCL4 (macrophage inflammatory protein β) are natural ligands for CCR5 and are inhibitors of HIV entry into cells (25). As mentioned above, we observed an increased CCR5 expression on CD4 T lymphocytes from coinfected patients so we assessed the circulating levels of RANTES/CCL5 and MIP1β/CCL4 in our groups of study. HIV monoinfected patients presented higher RANTES/CCL5 levels as compared with DENV/HIV coinfected patients (Figure 2 A). No difference was found in RANTES/CCL5 levels between DENV monoinfected and coinfected patients. Besides, we also showed that circulating RANTES/CCL5levels tended to be lower in acute DENV infected patients than in HD in agreement with our previous data (26). In addition, all groups DENV/HIV coinfection, DENV and HIV monoinfection presented increased levels of MIP1β/CCL4 as shown in Figure 2 B. 10

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Evaluation of inflammatory mediators in monoinfected DENV, coinfected DENV/HIV and monoinfected HIV The infection with HIV is characterized by chronic immune activation and is associated with

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inflammatory cytokines even during effective HAART (27). The cytokine storm play a key role in

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the pathogenesis of DENV infection (10). We next asked if the presence of DENV and HIV was

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associated with increased circulating levels of inflammatory mediators (cytokines and chemokines), since the inflammatory profile during DENV/HIV coinfection is still unknown. In general, concentrations of inflammatory mediators (IL-6, IL-8/CXCL8 and IP-10/CXL10) were increased in DENV patients (Figure 2 C, D and E, respectively) in agreement with our

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previous results (28-30).IL-8/CXCL8 and IL6 concentrations were significantly increased in all groups as compared with HD (Figure 2 C, D and E, respectively). Furthermore, a positive

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correlation was found between IL-8/CXCL8 and IL-6 levels in DENV positive patients, monoinfected and coinfected(r= 0.5601, ****p<0.00001). DENV monoinfected and coinfected showed higher circulating IP-10/CXCL10 levels

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compared with HD (Figure 2 E). Interestingly, the vast majority of patients who have developed

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more severe forms of dengue presented plasma levels of IP-10/CXCL10 above 10000 pg/ml. Moreover, circulating IP-10/CXCL10 levels were significantly lower in HIV monoinfected patients

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compared with DENV monoinfected and DENV/HIV coinfected (Figure 2 E). The imbalance of Th1/Th2 is observed during dengue and HIV infections (27, 31). No difference was found in the IFN-γ levels among groups, on the other hand IL-4 concentrations were

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significantly decreased in DENV/HIV coinfected group as compared with DENV monoinfected group (Figure 2 G).

IL-10 is anti-inflammatory cytokine and inhibits the synthesis of Th1 cytokines and also pro inflammatory cytokines (32).Circulating IL-10 levels were significantly higher in both DENV monoinfection and DENV/HIV coinfection groups, compared to HD In addition, HIV monoinfected patients showed decreased IL-10 levels compared with DENV monoinfected and DENV/HIV coinfected (Figure 2 H). Finally, there were no significant differences in the circulating levels of IFN-γ, IL-1RA and IL-17A among the different groups analyzed. We analyzed the cytokine patterns in relation to dengue severity. In this way, IL1RA levels were significant increased in severe DENV monoinfected patients compared with Dengue with no WS patients and HD [HD 13.9 (0.1-35.9), n=10, P<0.05; dengue with no WS 7.2 (0.1-27.3), n=25 P<0.01; and severe dengue 98.8 (28.9223.8), n=6]. 11

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Discussion DENV and HIV-1 infections are considered major health problems in Brazil. Nevertheless, few studies have documented DENV/HIV coinfection cases. Reported data so far suggests that

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DENV infection is associated with a transient reduction in HIV viral load (13) and no severe

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dengue outcome or HIV disease progression are observed during coinfection (14, 33, 34). However,

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it remains unknown the impact of DENV infection in the clinical outcome as well as in the immune response in individuals concomitantly infected with DENV and HIV. To our knowledge, the present study is the first to evaluate immunological parameters such as lymphocytes subsets and cytokine production in dengue patients having a HIV infection. Here we demonstrated that most coinfected

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patients presented mild clinical manifestations consistent with previous reports (33, 34). Elevated circulating CD8 T cell percentages in coinfected and higher circulating levels of inflammatory

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mediators were observed in coinfected patients suggesting that DENV infection in treated HIV infected individuals is a potential contributor of increased inflammatory response. At presentation, there was no significant difference between DENV/HIV and DENV

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patients according clinical symptoms and signs presented between groups, but coinfected

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DENV/HIV patients presented less severe bleeding, hemorrhagic manifestations and plasma leakage. Dengue infection is associated with thrombocytopenia (31) and is more pronounced in

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severe cases(35). On the other hand, primary HIV associate thrombocytopenia (PHAT) has been observed in HIV infected individuals and HAART seems to be effective in improving PHAT (36), but cases continue to be observed in the literature (37). Despite dengue infection, coinfected

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patients were less likely to have thrombocytopenia in agreement with their mildest clinical state. One probable explanation for a milder clinical outcome in coinfected cases is HIV inhibition by DENV and other pathogens such as GBV- C (GB virus type C) (38). Although, the data reported in the literature so far suggests that coinfection with dengue or other pathogens have suppressive effects on HIV infection, the mechanism responsible for inhibition is not fully understood. In this context, Watt et al suggested that DENV infection in HIV patient is associated with a reduction in viral loads (13). In fact, serum samples obtained during acute phase of infection were able to inhibit HIV replication in T CD4 lymphocytes in vitro suggesting that infection by DENV induces soluble factors capable to suppress the HIV viral replication. Similarly, The NS5 nonstructural protein of DENV-2 was also able to inhibit the HIV replication on T lymphocytes suggesting that coinfection with DENV and HIV may have inhibitory effects on HIV replication (16). Nonetheless, taking into account the few coinfected cases reported and in vitro studies, is not possible to ensure that coinfected individuals do not have an increased risk of a more rapid progression of AIDS in a later stage. In this context, recently, Pang et al. showed that, despite having similar clinical 12

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection manifestations when compared with DENV monoinfection, coinfected patients tended to be more severe than monoinfected patients even though under HAART regimens (39). Dengue infection is followed by leukopenia and reduced frequency of T lymphocytes

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including CD4 and CD8 subsets (30) and T cell activation play key role in the development of

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dengue severity (3). On the other hand, antiretroviral therapy induces rapid suppression of viral

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replication and a progressive restoration of CD4 T cells in HIV-infected individuals. However, a considerable proportion of treated individuals maintain high levels of circulating CD8T cells with persistently low CD4/CD8 ratios that have been associated with poor clinical outcomes (40) suggesting that CD8 T expansion is associated with morbidity and mortality. The quantitative

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differences of T lymphocytes subsets were assessed in the study groups. Although no significant difference was detected in CD4 T cells absolute counts, a significantly low CD4/CD8 ratio were

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observed in all individuals, monoinfected and coinfected as well as HIV monoinfected compared with healthy individuals. It was demonstrated that persistently low CD4/CD8 ratio< 1.0 during HAART may be a marker of persistent immune activation and inflammation. (23, 41).Despite the

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small number of samples tested by flow cytometry, we observed that most coinfected patients had

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CD4/CD8 rates<1 suggesting inadequate immune restoration. Our results are in agreement with others studies with others pathogens such as CMV and HCV that are a potential cause of deficient

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increase in CD4 and expansion on CD8 cells even after HAART (42, 43). Larger, well-conducted studies focused on immune restoration are needed in coinfected patients in order to confirm these results. In addition, a high frequency of double positive CD4+CD8+ T cells in DENV monoinfected

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and DENV/HIV coinfected were observed. Circulating CD4+CD8+ double positive T cells are found during diseases stages (44) and are highly reactive during HIV acute infection (45). CCR5 act as HIV coreceptor (46) and virus preferentially infects memory cells, probably due CCR5 expression on these cells (47). During HIV infection the CCR5 expression is frequently increased, mainly in CD4 T subset and the CCR5 levels tended to normalize during first months of therapy (48, 49). Our study found that CD4 cells expressing CCR5 were significantly increased in coinfected patients. Since all coinfected patients were under HAART and with undetectable viral loads the increasedCD4CCR5 expression could be partly attributed to DENV infection. In fact, DENV monoinfected patients also presented a higher CCR5 expression on CD8 T cells in agreement to our previous data (26).It was largely demonstrated that acute DENV infection is characterized by intense viral replication and activation of innate and adaptive immunity culminating in viral clearance and a long-term memory cells. Although T lymphocytes are not the main sources of viral replication, DENV may act indirectly in these cells and induce cellular activation, migration and susceptibility to apoptosis (9, 50) 13

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Chemokines such as RANTES/CCL5 and MIP1β/CCL4 are natural ligands for CCR5 and have been shown to be effective inhibitors of HIV(25). We also measured circulating levels of RANTES/CCL5 and MIP1β/CCL4 and analyzed the relationship between these ligands and CCR5

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expression. No association between chemokine levels and coreceptor expression was found,

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probable due limited number of cellular samples available for the study. However, DENVHIV

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coinfected patients presented decreased levels of RANTES compared to HIV monoinfection. Similarly, we demonstrated decreased circulating levels of RANTES/CCL5 during acute phase of dengue and a high expression on liver tissues of fatal cases, suggesting the migration of lymphocytes to inflamed tissues (26). Interestingly, the study by Hattori et al. showed that

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RANTES/CCL5 levels were significantly lower in coinfected individuals with HIV and GBV-C virus than in uninfected (17). Consistent with previous study by Kamat et al. that observed elevated

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levels of MIP1β/CCL4 in treated HAART HIV individuals (51), we observed that all groups studied presented increased levels ofMIP1β/CCL4 as compared with controls. Interestingly, MIP1β/CCL4 levels were associated with mild dengue cases (28).

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Our data suggest that coinfection can drive CD8 T cell expansion since only coinfected

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patients presented elevated CD8 T cells percentages, whereas HIV monoinfected had CD8 T percentages similar to healthy donors. In addition, we demonstrated that coinfected patients showed

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a cytotoxic profile as demonstrated by increased expression of CD107a in both T cell subsets and an increased CD107a expression among CCR5 positive T cells were found in both DENV monoinfected and DENV/HIV coinfected patients. During dengue infection an exacerbated

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activation of T cells is observed and both subsets showed a cytotoxic profile, more frequently in mild cases, suggesting an important role of these cells in disease severity (26, 50). Furthermore, it was demonstrated that a vigorous response by multifunctional CD8 T cells is associated with protection from dengue disease (52). An interesting study demonstrated functional differences in cell from DENV patients relative to severity, since that cells from severe patients were mainly inflammatory and produced cytokines such as IFN-γ and TNF-α, but were not cytotoxic (53). These data may explain, in part, why the vast majority of coinfected patients develop the mild form of dengue fever. Th1/Th2 cytokines maintain homeostasis and Th1/Th2 imbalance leads to immune disorders and diseases (54). In fact, Th2 cytokines responses are associated with immune suppression and progression of HIV infection, but this imbalance is adjusted by HAART(27). A Th1/Th2 imbalance has been associated with disease severity of dengue, but the published data differ among authors. Kadhiravan et al, observed an association of Th1 immune response with disease severity (55) while others demonstrated the opposite; so a Th2 immune response association with severe cases (56, 57). 14

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection We found increased levels of IL-4 as well as IFN-γ in severe dengue patients (28). Several factors could have biased these findings such as clinical classification; samples tested such as cells, plasma or serum, making hard the understanding about mechanism related to disease severity. Although no

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difference was observed in IFN-γ levels nor in the IFN-γ/IL4 ratio (data not shown), coinfected

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patients were associated with decreased IL-4 level suggesting a normalization of Th1/Th2 balance

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by HAART.

Inflammatory mediators play a critical role in the HIV pathogenesis as well as in dengue and it is widely accepted that inflammatory response and deregulated cytokine network are associated with development of dengue severity and no study so far has evaluated the cytokine patterns during

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coinfection (31, 58). In this way, circulating levels of IL-6 and IL-8/CXCL8 remained altered in all DENV positive patients, monoinfected and coinfected. Elevated IL-6 levels are a predictor of

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morbid outcomes in treated HIV patients (59, 60), and in dengue, IL-6 is frequently associated with disease severity (28, 61). Rathakrishnanet al. showed increased IL-8/CXCL8 levels in dengue patients with warning signs during deffervescence phase (62), while a recent study associated IL-

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8/CXCL8 with dengue severity (31, 63). Although we found altered circulating levels of IL-6 and

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IL-8/CXCL8 in both monoinfected and coinfected patients, we did not observed significant difference in relation to disease severity or clinical manifestations probably due small size of severe

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cases. Interestingly, a direct association between IL-6 and IL-8/CXCL8 circulating levels was found in both DENV mono and coinfected, suggesting an increased systemic inflammation in these patients.

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Increased IP-10/CXCL10 levels were previously detected in HIV patients on HAART (51, 64) and also in coinfection with hepatitis C virus (HCV) and HIV (65)suggesting a role in immune response to viral infections. In this way, IP-10/CXCL10 levels were increased in both DENV monoinfected and DENV/HIV coinfected patients. Futhermore, Dengue moninfected presented increased levels as compared withHIV monoinfected patients in agreement with our previous reports (26). More recently, severe cases of dengue were associated with IP10/CXCL10 circulating levels (29). The elevation of inflammatory mediators is usually accompanied by the increase in antiinflammatory factors. This balance have a key role in the outcomes of the infections. In this way, IL-10 is a potent anti-inflammatory cytokine (66) and a potential biomarker predictor for development of disease severity (29, 67). During dengue infection, circulating IL-10 levels have been associated with severe dengue and thrombocytopenia (29, 30). In addition, decreased levels are observed with therapy (68) as well as elite controllers had IL-10 levels comparable to HIV negative individuals (69). Although the IL-10 levels were increased in both groups mono and coinfected, we did not see significant difference among them or even between mild or severe cases 15

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection of dengue. As an anti-inflammatory cytokine, IL-10 may be important to control the inflammatory response and probably can increase the effectiveness of HAART. In summary, these observations attest to the presence of intense inflammatory response and immune activation in coinfected

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patients, in the absence of HIV viremia.

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Several factors can be attributed as cause of increased inflammation and immune activation for

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example, bacterial translocation. Reduced integrity of the mucosal epithelium is frequently observed during chronic HIV infection and causes bacterial translocation. Bacterial translocation has a key role in the chronic inflammation and high levels of lipopolysaccharide (LPS) levels in HIV infection and in coinfection with HCV are associated with increased inflammatory response

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(70). Recently, increased levels of LPS were found in dengue patients and associated with disease severity (71).

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There are some limitations in this study. The small sample size of DENV/HIV coinfection hampered statistical analyses. We only have patients on antiretroviral therapy with viral loads < 50 copies and thus we could not evaluate the viral loads over course of DENV infection. A prospective

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HIV and DENV viral loads assement in different phases of dengue infection should be done in

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future studies. However, it is important to known how coinfected patients behave in terms of clinical outcomes and immune response. Further studies, with larger sample sizes, are warranted to

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extend these findings. IL-1RA, IL17 and VEGF circulating levels were evaluated here and no significant difference was detected among groups of study. However, IL1RA levels were significantly increased in severe dengue patients in agreement with previous results (72). It is

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important to emphasize that the discrepancies observed in this study with respect to cytokine response observed in DENV monoinfection compared to literature data may be in part due to differences in the cytokine assays used, timing of sample collection and to genetic susceptibility of the patient or to the serotype of DENV infection and HAART regimens applied. In general, acute viral diseases can cause an accelerated progression of HIV, but our study corroborates previous data in which most DENV/HIV coinfected patients did not progress to a more severe disease. Importantly, activation of T lymphocytes and higher circulating levels of inflammatory makers were observed during coinfection even when HIV viral load is suppressed under HAART. The importance of increased inflammation in HIV infected individuals lies in its associations with comorbid and mortality. Our understanding about lymphocyte activation and cytokine production during DENV/HIV coinfection as well as the knowledge about immunological mechanisms involved with dengue severity in HIV patients is still limited and several important issues need to be clarified. However, we provided here novel data that may open insights into 16

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection immune response and clinical manifestations in individuals simultaneously infected with DENV and HIV that are crucial for prevention of severe cases of dengue in people at risk areas.

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None of the authors has any conflict of interest to disclose.

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Conflict of interest

Acknowledgments:

We gratefully thank Dra Ana Rita Motta Castro and her team, for their help and assistance with patient recruitment and sample collection; to Daniela Beghini for the help in the FACS CyAn

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facility; and MSc Márcio Cipitelli for assembly and resolution of figures. We are indebted to the Flavivirus Laboratory team at FIOCRUZ-RJ, especially Jaqueline Bastos Simões and MSc

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Fernanda Bruycker. We also would like to thank the Institute of Clinical Research Evandro Chagas for laboratory diagnosis of dengue and coinfected cases. We gratefully acknowledge the voluntarily participation from patients and healthy individuals in this study. This work was financially

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supported by IOC and PAPES/FIOCRUZ, CNPq/DECIT and FAPERJ.

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Figures legends Figure 1. Up regulation of CCR5 on T cells from patients mono or coinfected with DENV or HIV. PBMCs from healthy donors or from DENV monoinfetcted, DEN/HIV coinfected and HIV monoinfected patients were isolated and labeled as described in Materials and Methods section. Scatter dot plot plots represent data from PBMCs in healthy donors, DENV, DENV/HIV and HIV analyzed by flow cytometry for CCR5 and CD107a expression among CD4 and CD8 lymphocytes subsets. Groups of study: Healthy donor (62), DENV monoinfected patient, DENV/HIV coinfected and HIV monoinfected. 21

ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection (A) Cellular flow cytometry profile showing size and granulosity. (B) Representative dot plots showing the percentages of double CD4+CCR5+ cells subsets in HD, DENV, DENV/HIV and HIV, respectively.

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(C) All events analyzed were gated first within lymphocyte region and then on a CD4 +

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subpopulation.

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(D) Representative histograms of CCR5 expression among CD4 T lymphocytes between groups of study.

(E and F) Frequencies of CCR5; (G and H) CD107a; (I and J) and CD107a+CCR5+ or CCR5- are shown.

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Horizontal bars indicate the median values, interquartile range for each group of study (HD n = 10; DENV n= 16; DENV/HIV n=7; HIV n=14). Points painted by white and mixed colors in the graphs

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represent DENV monoinfected patients with severe and with WS, respectively. Statistical significance was assessed using the Kruskal-wallis test followed by Dunn’s multiple comparison test. Differences between patients and controls are given above the figures. * p<0,05; **p<0.01;

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***p<0,001.

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Figure 2. Circulating levels of cytokine and chemokines in patients mono or coinfected with DENV and HIV during dengue acute disease. The soluble factors were measured by immunoassays. Scatter plot graphs compared levels of cytokines and chemokines among groups of

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study. Horizontal bars indicate the median values and interquartile range for each group studied (HD; DENV; DENV/HIV; HIV). A-K. Circulating levels of cytokines RANTES/CCL5, MIP1β /CCL4, IL-6, IL-8/CXCL8, IP-10/CXCL10, IFN-γ, IL-4. IL-10, IL-1RA, IL-17A and VEGF in groups of study. Points painted by white and mixed colors in the graphs represent DENV monoinfected patients with severe and with WS, respectively. Statistical significance was assessed using the Kruskal-wallis test followed by Dunn’s multiple comparison test. Differences between patients and controls are given above the figures. * p<0,05; **p<0.01; ***p<0,001. Groups of study: healthy donors (HD), DENV monoinfected, DENV/HIV coinfected and HIV monoinfected patients.

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ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Table 1: Demographic and laboratory characteristics from the studied population with Dengue, HIV and coinfected.

Dengue and HIV coinfection

No WS

WS

Severe

No WS

n=10

n=41

n=17

n=16

8:2

23:18

11:6

7:9

40 (26-

34 (28-54)

34 (19-65)

Total number Gender,F:M

32 (21-41) a Age,Median (95% C.I.) b

50) 3 (2-4)

3 (3-5)

DENV infection, Primary:Secundary

-

4:15

0

c

-

43 (2797) (n=17) 45 (3199) (n=17) 42 (4143) (n=29) 153 (133189)*** (n=30)

62 (41142) (n=13) 59 (29138) (n=13) 40 (36-46) (n=15)

ALT, IU/L

N.D.

N.D.

a,d

Hematocrit,%

a

a,f

CD4/CD8 rate

+

+

2.0 (1.63.3) (n=10)

3945 (31505810) (n=29) 36 (1748) (n=9) 19 (1353) (n=9) 2.2 (0.33.5) (n=9)

1.8 (1.13.0) (n=10)

3.3 (1.85.5) (n=9)

22(15-27) (n=10)

a,f

CD4 CD8 ,%

252 (223333) (n=9)

6000 (52696300) (n=8) 43 (40-53) (n=10)

Leukocytes, a 3 mm counts

CD8, %

42 (37-46) (n=9)

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Platelets, a 3 3 10 /mm counts

CD4, %

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a,f

a,f

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a,d

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AST, IU/L

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HAART treatment

4 (2-4)

72 (38133)**** † (n=16)

3030 (23007400) (n=14) 23 (1942)*** (n=8) 23 (13-33) (n=8) 1.7 (0.61.9)** † (n=8) 3.4 (1.16.3) (n=8)

monoinfection Severe

-

n=13

n=7

n=1

n=14

5:10

3:4

1:0

6:8

41 (27-51)

41 (26-

46

47 (35-61)

50)

3 (2-4)

5 (1-6)

4

-

1:12

0:12

1:5

0:1

-

70 (35221) (n=13) 76 (33230) (n=13) 38 (3645)† (n=13) 43 (14126)**** ††††

15:15

7:7 48 (2272) (n=5) 46 (3347) (n=5) 46 (4348) (n=2) 216 (38388)£ (n=7)

1:1

14:14

158 (n=1)

-

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-

Illness day

HIV

WS

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donors

IP

Healthy

Dengue acute infection

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Controls Characteristics

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Group classification according to disease

§

(n=13) 5300 (193010900) (n=12) 33 (2243)* (n=5) 26 (21-42) (n=5) 1.6 (0.62.0)* (n=5) 4.2 (1.15.4)* (n=5)

48 (28-97) (n=12) 68 (37-99) (n=12) 39 (36-42) (n=6) 177 (106214)**** (n=13)

3040 (25204410)*** (n=13) 22(1242)*** (n=8) 36 (2544)*** (n=8) 0.6 (0.317)*** (n=8) 5.7 (3.08.8)****

69 (n=1) 32 (n=1)

-

40 (37-43) (n=14)

23 (n=1)

231 (185-275) (n=14)

5130 (24908120) (n=7) 16 (n=1)

1057 (n=1)

6665 (5200-8400) (n=14)

-

32 (20-41)** (n=12)

19 (n=1)

-

28 (20-38) (n=12)

0.8 (n=1)

-

1.0 (0.5-2.0) * (n=12)

1.7 (n=1)

-

2.3 (1.9-3.8) (n=12)

¥ (n=8)

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C.I., 95% Confidence interval.;

b

Illness Day , days from the start of any symptoms until the moment when the patient was interviewed;

c

HAART, Highly Active Antiretroviral Therapy;

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Transaminases,reference values for Aspartate aminotransferase (AST): Female 31 IU/L, Male 37 IU/L; Alanine

e

N.D.,Not determined;

CD4 and CD8 T frequencieswere determined by flow cytometry analysis;

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f

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aminotransferase (ALT): Female 31 IU/L, Male 41 IU/L.

*P<0.05, **P<0.01***P<0.001, ****P<0.0001 represent statistical difference vs. Control. Mann-Whitney nonparametric Test was applied.

†P<0.05, †† P<0.01 ††† P<0.001, †††† P<0.0001represent statistical differencevs.No WS. Mann-Whitney

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nonparametric test was applied.

§P<0.05 represent statistical difference vs.WS. Mann-Whitney nonparametric test was applied. £P<0.05 represent statistical difference DENV WS vs.DENV/ HIV WS. Mann-Whitney nonparametric test was applied.

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¥P<0.05represent statistical difference DENV No WS vs.DENV/ HIV No WS. Mann-Whitney nonparametric test was

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ACCEPTED MANUSCRIPT Torrentes-Carvalho et al. Immunological parameters in dengue and HIV coinfection Table 2: Clinical features associated with severity of dengue in the mono and coinfected patients. Monoinfected DENV 17 (32.7%)

0.008**

Severe bleeding

0

14 (26.9%)

0.0960

Plasma leakage

1 (4.3%)

7 (13.5%)

0.2624

Pleural effusions

0

5 (9.6%)

0.3134

Severe Dengue

1 (4.3 %)

14 (26.9%)

0.0646

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Hemorrhagic manifestations

Coinfected + DENV HIV 1 (4.3 %)

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Statistical significance was assessed using Fisher`s exact test. Statistical significance is represented by ** (P<0.01).

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Highlights: 1. Diagnosis of severe dengue was less frequent in DENV/HIV coinfection; 2. Coinfected patients presented low CD4+ T cells% with increased in CCR5 expression; 3. An increased CD107a expression on both T subsets was observed during coinfection; 4. Chemokines IP-10 and IL-8 were significantly altered in plasma of coinfected patients; 5. Cytokines involved in dengue pathogenesis were also increased during coinfection.

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