Marcia Woda

Enhanced humoral and HLA‐A2‐restricted dengue virus‐specific T‐cell responses in humanized BLT NSG mice

Dengue is a mosquito‐borne viral disease of humans, and animal models that recapitulate human immune responses or dengue pathogenesis are needed to understand the pathogenesis of the disease. We recently described an animal model for dengue virus (DENV) infection using humanized NOD‐scid IL2rγnull mice (NSG) engrafted with cord blood haematopoietic stem cells. We sought to further improve this model by co‐transplantation of human fetal thymus and liver tissues into NSG (BLT‐NSG) mice. Enhanced DENV‐specific antibody titres were found in the sera of BLT‐NSG mice compared with human cord blood haematopoietic stem cell‐engrafted NSG mice. Furthermore, B cells generated during the acute phase and in memory from splenocytes of immunized BLT‐NSG mice secreted DENV‐specific IgM antibodies with neutralizing activity. Human T cells in engrafted BLT‐NSG mice secreted interferon‐γ in response to overlapping DENV peptide pools and HLA‐A2 restricted peptides. The BLT‐NSG mice will allow assessment of human immune responses to DENV vaccines and the effects of previous immunity on subsequent DENV infections.

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.

Dengue Virus Infection Differentially Regulates Endothelial Barrier Function over Time through Type I Interferon Effects

BACKGROUND The morbidity and mortality resulting from dengue hemorrhagic fever (DHF) are largely caused by endothelial barrier dysfunction and a unique vascular leakage syndrome. The mechanisms that lead to the location and timing of vascular leakage in DHF are poorly understood. We hypothesized that direct viral effects on endothelial responsiveness to inflammatory and angiogenesis mediators can explain the DHF vascular leakage syndrome. METHODS We used an in vitro model of human endothelium to study the combined effects of dengue virus (DENV) type 2 (DENV2) infection and inflammatory mediators on paracellular macromolecule permeability over time. RESULTS Over the initial 72 h after infection, DENV2 suppressed tumor necrosis factor (TNF)-alpha-mediated hyperpermeability in human umbilical vein endothelial cell (HUVEC) monolayers. This suppressive effect was mediated by type I interferon (IFN). By 1 week, TNF-alpha stimulation of DENV2-infected HUVECs synergistically increased cell cycling, angiogenic changes, and macromolecule permeability. This late effect could be prevented by the addition of exogenous type I IFN. CONCLUSIONS DENV infection of primary human endothelial cells differentially modulates TNF-alpha-driven angiogenesis and hyperpermeability over time. Type I IFN plays a central role in this process. Our findings suggest a rational model for the DHF vascular leakage syndrome.

Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-gamma-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice.

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγnull mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.

Neonatal BCG vaccination is associated with enhanced T-helper 1 immune responses to heterologous infant vaccines.

Neonatal Bacille Calmette Guérin (BCG) vaccination has been reported to have beneficial effects beyond preventing infantile tuberculous meningitis and miliary disease. We hypothesized that BCG vaccine given at birth would enhance T-helper 1 (Th1) immune responses to the first vaccines given later in infancy. We conducted a nested case-control study of neonatal BCG vaccination and its heterologous Th1 immune effects in 2–3 months old infants. BCG vaccination at birth was associated with an increased frequency of interferon-γ (IFN-γ) producing spot-forming cells (SFC) to tetanus toxoid 2–3 months later. The frequency of IFN-γ producing SFC to polioviruses 1–3 also trended higher among infants who received BCG vaccination at birth. The frequency of IFN-γ+/tumor necrosis factor-α (TNF-α)+CD45RO+CD4+ T-cells upon stimulation with phorbol myristate acetate (PMA)/Ionomycin was higher in 2–3 months old infants who received BCG vaccination at birth compared to those who did not. The circulating frequency of forkhead box P3 (FoxP3)+ CD45RO+ regulatory CD4+ T-cells also trended lower in these infants. Neonatal BCG vaccination is associated with heterologous Th1 immune effects 2–3 months later.

Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells

Killer immunoglobulin‐like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR–HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self‐limited viral infections. During our investigation of CD8+ T cell responses to a conserved HLA‐B57‐restricted epitope derived from dengue virus (DENV) non‐structural protein‐1 (NS1), we observed substantial binding of the tetrameric complex to non‐T/non‐B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long‐standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56dim NK cells, which are known to express KIRs. Using depletion studies and KIR‐transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA‐B57+ subjects with acute DENV infection revealed marked activation of NS1 tetramer‐binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR–HLA interactions in the modulation of disease outcomes.

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection

Variation in the sequence of T‐cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T‐cell responses during second heterologous infections. We identified a highly conserved, novel, HLA‐B57‐restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57‐NS126–34‐specific CD8+ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer‐positive T‐cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57‐NS126–34‐specific and other DENV epitope‐specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope‐specific CD8+ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen‐specific T‐cell activation.

Plasmacytoid dendritic cell interferon-α production to R-848 stimulation is decreased in male infants

BackgroundSex differences in response to microbial infections, especially viral ones, may be associated with Toll-like receptor (TLR)-mediated responses by plasmacytoid dendritic cells (pDCs).ResultsIn this study, we identified sex differences in human infant pDC interferon-α production following challenge with the TLR7/8 agonist R-848. Male pDC responses were significantly lower than those of females during early infancy. This difference may be attributed to the androgen surge experienced by males during the early infancy period. Pretreatment of human pDCs with dihydrotestosterone produced a significant reduction in interferon-α production following R-848 challenge.ConclusionsAndrogen-mediated regulation of pDC TLR7-driven innate immune responses may contribute to the observed sex differences in response to infections during early infancy.

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections

BackgroundDeclining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years.ResultsVACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire.ConclusionsTLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.