Heather Friberg

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research. Methodology/Principal Findings We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγnull) mice engrafted with human hematopoietic stem cells. Human CD45+ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγnull mice with HLA-A2+ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353(111–119), NS4b 2423(181–189), and NS4a 2148(56–64). Conclusions/Significance This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.

Cross-reactivity and expansion of dengue-specific T cells during acute primary and secondary infections in humans

Serotype-cross-reactive memory T cells responding to secondary dengue virus (DENV) infection are thought to contribute to disease. However, epitope-specific T cell responses have not been thoroughly compared between subjects with primary versus secondary DENV infection. We studied CD8+ T cells specific for the HLA-A*1101-restricted NS3133 epitope in a cohort of A11+ DENV-infected patients throughout acute illness and convalescence. We compared the expansion, serotype-cross-reactivity, and activation of these cells in PBMC from patients experiencing primary or secondary infection and mild or severe disease by flow cytometry. Our results show expansion and activation of DENV-specific CD8+ T cells during acute infection, which are predominantly serotype-cross-reactive regardless of DENV infection history. These data confirm marked T cell activation and serotype-cross-reactivity during the febrile phase of dengue; however, A11-NS3133-specific responses did not correlate with prior antigenic exposure or current disease severity.

Memory CD8 + T cells from naturally-acquired primary dengue virus infection are highly cross-reactive

Cross‐reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T‐cell response to heterologous serotypes, we isolated HLA‐A∗1101‐restricted epitope‐specific CD8+ T‐cell lines from primary DENV‐immune donors. Cell lines exhibited marked cross‐reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross‐reactivity between the DENV‐1 and DENV‐3 epitope variants. In vitro‐stimulated T‐cell lines consistently revealed a hierarchical induction of MIP‐1β>degranulation>tumor necrosis factor α (TNFα)>interferon‐γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose‐dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP‐1β production. This is the first study to show significant DENV serotype‐cross‐reactivity of CD8+ T cells after naturally acquired primary infection. We also show qualitatively different T‐cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.

Impact of prior flavivirus immunity on Zika virus infection in rhesus macaques

Studies have demonstrated cross-reactivity of anti-dengue virus (DENV) antibodies in human sera against Zika virus (ZIKV), promoting increased ZIKV infection in vitro. However, the correlation between in vitro and in vivo findings is not well characterized. Thus, we evaluated the impact of heterotypic flavivirus immunity on ZIKV titers in biofluids of rhesus macaques. Animals previously infected (≥420 days) with DENV2, DENV4, or yellow fever virus were compared to flavivirus-naïve animals following infection with a Brazilian ZIKV strain. Sera from DENV-immune macaques demonstrated cross-reactivity with ZIKV by antibody-binding and neutralization assays prior to ZIKV infection, and promoted increased ZIKV infection in cell culture assays. Despite these findings, no significant differences between flavivirus-naïve and immune animals were observed in viral titers, neutralizing antibody levels, or immune cell kinetics following ZIKV infection. These results indicate that prior infection with heterologous flaviviruses neither conferred protection nor increased observed ZIKV titers in this non-human primate ZIKV infection model.

Differential In Vivo Clearance and Response to Secondary Heterologous Infections by H2b-Restricted Dengue Virus-Specific CD8+ T Cells

Cytotoxic T lymphocytes (CTL) are hypothesized to play a role in clearance during primary dengue virus (DENV) infections, and contribute to immunopathology during secondary heterologous infections in humans. We previously reported skewed T-cell responses to secondary DENV infection in BALB/c (H-2(d)) mice, reproducing characteristics of human DENV infection. To set the stage for using widely available transgenic and knockout mice, we extended these studies to identify DENV-specific T-cell responses in C57BL/6 (H-2(b)) mice. We identified dominant CD8+ T-cell responses to H-2D(b)-restricted epitopes on the DENV NS4a (aa 249-265) and NS5 (aa 521-537) proteins. High frequencies of IFN-γ- and TNF-α-producing T cells directed at both epitopes were detected following primary infection with all four DENV serotypes, and were augmented by secondary DENV infections. In vivo cytotoxicity assays demonstrated rapid clearance of target cells pulsed with the NS4a peptide; in contrast, NS5 peptide-pulsed target cells were poorly cleared in vivo. These data characterize two H-2(b)-restricted T-cell epitopes displaying divergent in vivo function. These results should facilitate further studies of the in vivo effects of DENV-specific T cells, including the use of genetically modified mouse strains.

Analysis of Human Monoclonal Antibodies Generated by Dengue Virus-Specific Memory B Cells

Dengue, caused by the four serotypes of dengue virus (DENV), represents an expanding global health challenge. The potential for serotype-cross-reactive antibodies to exacerbate disease during a secondary infection with a heterologous DENV serotype has driven efforts to study human DENV-specific antibodies. Most DENV-specific antibodies generated in humans are serotype-cross-reactive, weakly neutralizing, and directed against the immature pre-membrane (prM), envelope (E), and nonstructural 1 (NS1) proteins. To broaden the characterization of human DENV-specific antibodies, we assessed B-cell responses by ELISpot assays and isolated B cells from the peripheral blood of a human subject with previous DENV infection. Forty-eight human IgG monoclonal antibodies (hMAbs) were initially characterized by their potential to bind to an inactivated lysate of DENV-infected cells. Subsequently, most DENV-specific hMAbs were found to bind soluble, recombinant E protein (rE). Two hMAbs were unable to bind rE, despite strongly binding to the DENV-infected cell lysate. Further analyses showed that these two hMAbs bound conformation-dependent, reduction-sensitive epitopes on E protein. These data shed light on the breadth of DENV-specific hMAbs generated within a single immune donor.

Safety and Immunogenicity of a Dengue Virus Serotype-1 Purified-Inactivated Vaccine: Results of a Phase 1 Clinical Trial

We describe the results from a human clinical trial of a dengue virus serotype-1, purified-inactivated vaccine (DENV-1 PIV) adjuvanted with aluminum hydroxide. This first-in-man, Phase 1, open-label clinical trial consisted of two groups of flavivirus-naïve healthy adult volunteers that received two intramuscular vaccine doses of either 2.5 μg or 5 μg of DENV-1 PIV administered on days 0 and 28. Following vaccination, both vaccine doses exhibited an acceptable safety profile with minimal injection site and systemic reactions. By study day 42, 2 weeks following the second vaccine dose, all volunteers in both vaccine groups developed serum-neutralizing antibodies against DENV-1. Additional testing using an enzyme-linked immunosorbent assay demonstrated induction of a humoral immune response following both vaccine doses. The DENV-1 PIV was safe and immunogenic in a small number of volunteers supporting development and further testing of a tetravalent DENV PIV formulation.

Immunopathogenesis versus protection in dengue virus infections

Dengue viruses (DENV) are mosquito-borne viruses that cause significant morbidity. The existence of four serotypes of DENV with partial immunologic cross-reactivity creates the opportunity for individuals to experience multiple acute DENV infections over the course of their lifetimes. Research over the past several years has revealed complex interactions between DENV and the human innate and adaptive immune systems that can have either beneficial or detrimental influences on the outcome of infection. Further studies that seek to distinguish protective from pathological immune responses in the context of natural DENV infection, as well as clinical trials of candidate DENV vaccines, are an important part of efforts to control the global impact of this re-emerging viral disease.

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

BACKGROUND The development of reagents to identify and characterize antigen-specific B cells has been challenging. METHODS We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. RESULTS In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. CONCLUSIONS AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination.

Analysis of cell-mediated immune responses in support of dengue vaccine development efforts.

Dengue vaccine development has made significant strides, but a better understanding of how vaccine-induced immune responses correlate with vaccine efficacy can greatly accelerate development, testing, and deployment as well as ameliorate potential risks and safety concerns. Advances in basic immunology knowledge and techniques have already improved our understanding of cell-mediated immunity of natural dengue virus infection and vaccination. We conclude that the evidence base is adequate to argue for inclusion of assessments of cell-mediated immunity as part of clinical trials of dengue vaccines, although further research to identify useful correlates of protective immunity is needed.