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Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice

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Abstract

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT−/− mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.

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Acknowledgments

We thank Ilma Marçal, Gilvânia Ferreira da Silva Santos and Franckcinéia Assis (ICB/UFMG) for technical assistance. Instituto Nacional de Ciência e Tecnologia em Dengue (INCT-Dengue); PRONEX em Dengue (Ministério da Saúde), Brazil; Conselho Nacional de Ciência de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). VVC and CTF are supported by the “Science without Borders” program.

Conflict of interest

The authors have no conflicting financial interests.

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Authors and Affiliations

  1. Laboratório de Interação Microrganismo-Hospedeiro, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Vivian V. Costa, Caio T. Fagundes, Deborah F. Valadão, Thiago V. Ávila, Daniel Cisalpino, Rebeca F. Rocha, Lucas S. Ribeiro, Fernando R. Ascenção & Danielle G. Souza

  2. Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Vivian V. Costa, Caio T. Fagundes, Deborah F. Valadão, Thiago V. Ávila, Daniel Cisalpino, Rebeca F. Rocha, Lucas S. Ribeiro, Fernando R. Ascenção, Celso M. Q. Junior, Tarcília A. Silva, Mauro M. Teixeira & Danielle G. Souza

  3. Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore

    Vivian V. Costa

  4. Inflammation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland

    Caio T. Fagundes

  5. Laboratório de Farmacologia Cardiovascular, Departamento de Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Lucas M. Kangussu & Daniela Bonaventura

  6. Departamento de Patologia Oral, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Celso M. Q. Junior & Tarcília A. Silva

  7. Laboratório de Virologia Básica e Aplicada, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

    Ruiz G. Astigarraga

  8. Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife, PE, Brazil

    Frederico L. Gouveia & Ana Cristina L. Leite

  9. Fundação de Hematologia e Hemoterapia de Pernambuco - HEMOPE, Recife, PE, Brazil

    Frederico L. Gouveia, Divaldo de Almeida Sampaio & Ana Cristina L. Leite

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  1. Vivian V. Costa
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  17. Mauro M. Teixeira
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  18. Danielle G. Souza
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Corresponding author

Correspondence to Danielle G. Souza.

Electronic supplementary material

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430_2014_334_MOESM1_ESM.tif

Figure S1: μMT −/− mice develop severe liver injury after primary DENV-3 inoculation. WT (n = 5) and μMT−/− (n = 7) mice were inoculated with 100PFU of DENV-3 (i.p) and seven days later, mice were culled and the liver collected for performing histopathological analysis. Liver tissues were formalin-fixed and processed into paraffin sections and were stained with hematoxylin and eosin. Histopathological scores of each mouse were performed. Representative images of each group of mice are shown (Scale Bar - 400 μm). All results are expressed as mean ± SEM and are representative at least two experiments. * for P < 0.05 when compared to WT-infected mice. NI—non-infected. HS – hepatocyte swelling. D – degeneration. N – necrosis. H – hemorrhage. OS – overall score (TIFF 11435 kb)

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Figure S2: Subneutralizing titers of DENV-3-immune serum decreases survival time of DENV-3-inoculated mice and aggravate liver injury in DENV-3-inoculated mice. (A) WT mice (n = 8 mice per group) were administered with naïve serum or anti-DENV-3 serum (collected on day 49) and inoculated with 100PFU of DENV-3 (i.p) and lethality rates was evaluated every 12 h during 14 days. Results are expressed as  % of survival. In B, WT mice (n = 5 per group) treated with naïve or anti-DENV-3 serum were inoculated with 100PFU of DENV-3 (i.p), and seven days after infection, mice were culled and tissue collected for the histopathological analysis. Liver of control and DENV-3-infected mice were collected, formalin-fixed and processed into paraffin sections. Liver sections were stained with hematoxylin and eosin, and histopathological scores of each mouse were performed. Representative images of each group of mice are shown (Scale Bar - 400 μm). All results are expressed as mean ± SEM and are representative at least two experiments. # for P < 0.05 when compared to WT-infected naïve serum treated mice. Dpi—days post-infection. HS—hepatocyte swelling. D—degeneration. N—necrosis. H—hemorrhage. OS—overall score (TIFF 11830 kb)

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Figure S3: DENV-induced liver injury in 4G2-treated mice depends on mAb dose. WT mice (n = 6 per group) were administered with different doses of the mouse monoclonal antibody—4G2 (IgG2a anti-Envelope protein, pan-flavivirus-reactive) or with an isotype control antibody with irrelevant specificity followed by inoculation of 100PFU of DENV-3 (i.p). Seven days later, mice were culled tissue collected for the histopathological analysis. Liver of control and DENV-3-infected mice were collected, formalin-fixed and processed into paraffin sections. Liver sections were stained with hematoxylin and eosin, and histopathological scores of each mouse were performed. Representative images of each group of mice are shown (Scale Bar - 400 μm). All results are expressed as mean ± SEM and are representative at least two experiments. # for P < 0.05 when compared to isotype control DENV-3-infected mice. ** for P < 0.05 when compared to 4G2 (15 μg) DENV-3-infected mice. HS—hepatocyte swelling. D—degeneration. N—necrosis. H—hemorrhage. OS—overall score (TIFF 12555 kb)

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Figure S4. In vivo blockade of FcγRs impairs the liver injury induced by the treatment with subneutralizing doses of anti-DENV mAbs in DENV-infected mice. WT mice (n = 5 per group) were administered with 15 μg of the mouse monoclonal antibody—4G2 (IgG2a anti-Envelope protein, pan-flavivirus-reactive) or with an isotype control antibody with irrelevant specificity followed by inoculation of 100PFU of DENV-3 (i.p). Another two groups received the anti-DENV 4G2 mAb together with the Fc-blocking antibody (clone 2.4G2) or were administered with the isotype control mAb and the Fc-blocker. Seven days later, mice were culled tissue collected for performing the histopathological analysis. Liver of control and DENV-3-infected mice were collected, formalin-fixed and processed into paraffin sections. Liver sections were stained with hematoxylin and eosin, and histopathological scores of each mouse were performed. Representative images of each group of mice are shown (Scale Bar - 400 μm). All results are expressed as mean ± SEM and are representative at least two experiments. # for P < 0.05 when compared to isotype control or isotype control + 2.4G2 DENV-3-infected mice. ** for P < 0.05 when compared to 4G2 DENV-3-infected mice. ND—non-detected. HS—hepatocyte swelling. D—degeneration. N—necrosis. H—hemorrhage. OS—overall score (TIFF 12766 kb)

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Figure S5: Mice deficient for the type I (IFN-α/β) receptors (A129 −/− ) are very susceptible to primary DENV infection. (A) WT and A129 −/− mice (n = 8 mice per group) were inoculated with 100PFU of DENV-2 (i.p), and lethality rates were evaluated every 12 h during 12 days. Results are expressed as  % of survival. In B-G, WT (n = 5) and A129 −/− mice (n = 5-10) were inoculated with 100PFU of DENV-2 (i.p), and three, five or seven days after infection, mice were culled and blood and tissue collected for the following analysis: (B-C) Viral loads recovered from spleen or blood by plaque assay in Vero cells. Results are shown as the log of PFU per g of spleen or PFU per mL of blood, respectively. (D) Number of platelets, shown as platelets X 103/μl of blood. (E) Hematocrit, shown as  % volume occupied by red blood cells. (F) Concentrations of TNF-α in serum, quantified by ELISA. Results are shown as pg per mL (serum). (G) ALT activity determination in plasma of control and DENV-2-infected mice is shown as U/dL of plasma. All results are expressed as mean ± SEM (except for B-C, expressed as median) and are representative of at least two experiments. * for P < 0.05 when compared to control uninfected mice. # for P < 0.05 when compared to WT-infected mice. NI—non-infected. dpi—days post-infection. ND—non-detected (TIFF 2917 kb)

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Figure S6: Treatment of mice with IVIG containing non-neutralizing levels of α-DENV antibodies enhances liver injury after DENV-3 inoculation. Vehicle and α-DENV IVIG-treated mice—dose of 100 mg/kg (n = 5 per group)—were inoculated with 100PFU of DENV-3 (i.p), and seven days later, mice were culled and the liver collected for performing histopathological analysis. Liver of control and DENV-3-infected mice were collected, formalin-fixed and processed into paraffin sections. Liver sections were stained with hematoxylin and eosin, and histopathological scores of each mouse were performed. Representative images of each group of mice are shown (Scale Bar - 400 μm). Results are expressed as mean ± SEM and are representative at least two experiments. * for P < 0.05 when compared to vehicle-treated and DENV-3-infected mice. NI—non-infected. HS—hepatocyte swelling. D—degeneration. N—necrosis. H—hemorrhage. OS—overall score (TIFF 10166 kb)

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Costa, V.V., Fagundes, C.T., Valadão, D.F. et al. Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice. Med Microbiol Immunol 203, 231–250 (2014). https://doi.org/10.1007/s00430-014-0334-5

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