Altered monocyte response to the dengue virus in those with varying severity of past dengue infection.

OBJECTIVE: We sought to investigate the differences in monocyte immune responses to the dengue virus (DENV) in those who previously had either severe disease (past SD) or non-severe dengue (past NSD) following a secondary dengue infection. METHOD: Monocytes from healthy individuals who had either past SD (n = 6) or past NSD (n = 6) were infected at MOI one with all four DENV serotypes following incubation with autologous serum. 36-hours post infection, levels of inflammatory cytokines and viral loads were measured in the supernatant and expression of genes involved in viral sensing and interferon signaling was determined. RESULTS: Monocytes of individuals with past SD produced significantly higher viral loads (p = 0.0426 and cytokines (IL-10 p = 0.008, IL-1ß p = 0.008 and IL-6 p = 0.0411) when infected with DENV serotypes they were not immune to, compared to those who has past NSD. Monocytes of individuals with past SD also produced significantly higher viral loads (p = 0.022) and cytokines (IL-10 p < 0.0001, IL-1ß < 0.0001 and IL-6 p < 0.0001) when infected with DENV serotypes they were previously exposed to, despite the monocytes being infected in the presence of autologous serum. A significant upregulation of NLRP3 (p = 0.005), RIG-I (0.0004) and IFNB-1 (0.01) genes were observed in those who had past SD compared to past NSD when infected with non-immune DENV serotypes. CONCLUSION: Monocytes from those with past SD appear to show marked differences in viral loads, viral sensing and production of inflammatory mediators in response to the DENV, when compared to those who experienced past NSD, suggesting that initial innate immune responses may influence the disease outcome.

Identification of serotype-specific T cell responses to highly conserved regions of the dengue viruses.

Determining previous infecting dengue virus (DENV) serotypes has been difficult due to highly cross-reactive immune responses from previous DENV infections. Determining the correlates of serotype-specific immune responses would be crucial in understanding dengue transmission in the community and would also help to determine the correlates of protective immune responses. Therefore, we set out to define highly conserved, serotype-specific regions of the DENVs. Serotype-specific and highly conserved regions of the four DENV serotypes were identified using Basic Local Alignment Search Tool (BLAST) searches and custom perl scripts. Using ex-vivo and cultured enzyme-linked immunospot (ELISPOT) assays, we identified serotype-specific T cell epitopes within the four DENV serotypes in healthy adult donors from Sri Lanka. We identified T cell responses to 19 regions of the four DENV serotypes. Six peptides were from the NS2A region and four peptides were from the NS4A region. All immune donors responded to peptides of at least two DENV serotypes, suggesting that heterologous infection is common in Sri Lanka. Eight of 20 individuals responded to at least two peptides of DENV-4, despite this serotype not being implicated previously in any of the epidemics in Sri Lanka. The use of these regions to determine past and current infecting DENV serotypes will be of value to characterize further the dynamics of silent dengue transmission in the community. In addition, these T cell responses to these regions could be used to characterize DENV serotype-specific immune responses and thus possibly help us to understand the immune correlates of a protective immune response.

IE63-specific T-cell responses associate with control of subclinical varicella zoster virus reactivation in individuals with malignancies.

BACKGROUND: Reactivation of the varicella zoster virus (VZV) is more common in patients with malignancies; however, the molecular and cellular mechanisms underlying this susceptibility are unclear. METHODS: Using ex vivo interferon-gamma ELISpot assays, we set out to analyse VZV-specific immune responses in a large cohort of patients with malignancies. RESULTS: We observed that patients with malignancies had impaired VZV-specific T-cell responses, particularly in those with haematological malignancies and breast carcinoma. Immediate-early protein 63 (IE63)-specific T-cell responses were significantly impaired in those with subclinical VZV re-activation. CONCLUSIONS: Our results suggest that T-cell responses to IE63 are important in controlling VZV replication.

Immune evasion during varicella zoster virus infection of keratinocytes.

T cells are sensitized during varicella-zoster virus (VZV) infection and are important for control of viral spread and reactivation. In this report, we show that human keratinocytes infected with VZV inhibited upregulation of major histocompatibility complex (MHC) class I, MHC class II and intercellular adhesion molecule-1 after interferon (IFN)-gamma treatment. The ability of keratinocytes to upregulate MHC class I in response to IFN-alpha, tumour necrosis factor (TNF)-alpha and Toll-like receptor (TLR)-3 ligand was also diminished upon VZV infection. VZV-infected keratinocytes treated with IFN-gamma had significantly reduced capacity to stimulate antigen-specific T cells compared with uninfected cells. Interference with IFN-alpha, TNF-alpha, IFN-gamma and TLR-3 signalling in keratinocytes by VZV may contribute to immune evasion of the adaptive immune response.

Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors.

Varicella zoster viru (VZV)-specific T cell responses are believed to be vital in recovery from primary VZV infection and also in the prevention of viral reactivation. While glycoprotein E (gE) is the most abundant and one of the most immunogenic proteins of the virus, there are no data addressing potential T cell epitopes within gE, nor the phenotype of specific T cells. Using interferon gamma enzyme-linked immunospot assays and intracellular cytokine assays, we identified gE-specific immune responses in 20 adult healthy immune donors which were found to be dominated by the CD4+ subset of T cells. We characterized three immune dominant epitopes within gE restricted through DRB1*1501, DRB1*07 and DRB4*01, and used DRB1*1501 class II tetrameric complexes to determine the ex vivo frequency and phenotype of specific T cells. In healthy immune donors, the cells were largely positive for CCR7, CD28 and CD27, but expressed variable CD62L and low levels of cutaneous lymphocyte associated antigen with evidence of recent activation. In summary, we show that circulating gE-specific CD4+ T cells are detected at a relatively high frequency in healthy immune donors and show evidence of recent activation and mixed central and effector memory phenotype. These data would be compatible with frequent exposure to replicative cycle antigens in healthy donors and are consistent with a role for gE-specific CD4+ T cells in the control of viral replication.