Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

BACKGROUND: Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. METHODS: We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. RESULTS: We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. CONCLUSIONS: These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

There is growing interest in understanding antibody (Ab) function beyond neutralization. The non-structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody-dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody-dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV-specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus-like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1-specific Ab-secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1-expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1-expressing CEM-NKR cell lines using ZIKV-immune but not ZIKV-naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM-NKR cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells.

Multiplexed FluoroSpot for the Analysis of Dengue Virus- and Zika Virus-Specific and Cross-Reactive Memory B Cells.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

Activation of Peripheral T Follicular Helper Cells During Acute Dengue Virus Infection.

Background: Follicular helper T cells (TFH) are specialized CD4 T cells required for B-cell help and antibody production. Methods: Given the postulated role of immune activation in dengue disease, we measured the expansion and activation of TFH in the circulation (peripheral TFH [pTFH]) collected from Thai children with laboratory-confirmed acute dengue virus (DENV) infection. Results: We found significant expansion and activation of pTFH subsets during acute infection with the highest frequencies of activated pTFH (PD1hi pTFH and PD1+CD38+ pTFH) detected during the critical phase of illness. Numbers of activated pTFH were higher in patients with secondary compared with primary infections and in patients with more severe disease. We also found a positive correlation between the frequencies of activated pTFH and the frequencies of plasmablasts. Conclusions: To our knowledge, this is the first ex vivo analysis of pTFH activation during acute DENV infection. Overall, our study supports the model that pTFH contribute to disease evolution during the critical stage of illness.

Defining the role of NK cells during dengue virus infection.

In recent years, our understanding of the complex number of signals that need to be integrated between a diverse number of receptors present on natural killer (NK) cells and ligands present on target cells has improved. Here, we review the progress made in identifying interactions between dengue viral peptides presented on HLA Class 1 molecules with inhibitory and activating killer-like immunoglobulin receptors on NK cells, direct interactions of viral proteins with NK cell receptors, the involvement of dengue virus-specific antibodies in mediating antibody-dependent cell-mediated cytotoxicity and the role of soluble factors in modulating NK cell responses. We discuss findings of NK cell activation early after natural dengue infection, and point to the role that NK cells may play in regulating both innate and adaptive immune responses, in the context of our new appreciation of interactions of dengue virus with specific NK cell receptors. With a number of flavivirus vaccine candidates in clinical trials, how NK cells respond to attenuated dengue virus and subunit protein vaccine candidates and shape adaptive immunity will need to be considered.

Regulation and Function of NK and T Cells During Dengue Virus Infection and Vaccination.

The focus of this review is to discuss findings in the last 10 years that have advanced our understanding of human NK cell responses to dengue virus. We will review recently identified interactions of activating and inhibitory receptors on NK cells with dengue virus, human NK responses to natural dengue infection and highlight possible interactions by which NK cells may shape adaptive immune responses. T cell responses to natural dengue infection will be reviewed by Laura Rivino in Chap. 17 . With the advent of numerous dengue vaccine clinical trials, we will also review T and NK cell immune responses to dengue virus vaccination. As our understanding of the diverse functions of NK cell has advanced, it has become increasingly clear that human NK cell responses to viral infections are more complicated than initially recognized.

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.

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-NS1(26-34) -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-NS1(26-34) -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.

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

BACKGROUND: Declining 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. RESULTS: VACV- 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. CONCLUSIONS: TLs 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.

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 IL2rγ(null) 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.

Defective pro-IL-1ß responses in macrophages from aged mice.

BACKGROUND: Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1ß production by macrophages from aged mice. We examined the production of pro-IL-1ß and its conversion into IL-1ß as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6-8 weeks) and aged (18-24 months) C57BL/6 mice. FINDINGS: Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1ß after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines. CONCLUSIONS: Lower levels of IL-1ß production are a result of slower and lower overall production of pro-IL-1ß in macrophages from aged mice.

Understanding the contribution of cellular immunity to dengue disease pathogenesis.

SUMMARY: Dengue viruses (DENV) are the mosquito-borne viruses of greatest global public health importance. DENV circulate as four serotypes with significant immunologic cross-reactivity that does not provide protection from secondary infection with heterologous serotypes. The strong association of severe dengue illness, dengue hemorrhagic fever (DHF), with heterologous secondary infection and high cytokine levels has led to a prevailing view that DHF is immunologically mediated. In vitro studies of DENV-specific T lymphocytes, clinical studies of acute DENV infection, and immunologic studies in mouse models have provided evidence that in heterologous secondary DENV infection, there is preferential activation of memory T lymphocytes with lower avidity for the infecting virus ('original antigenic sin') resulting in altered T-cell functional responses. In the setting of host genetic predisposition and high level viremia, with resulting high antigenic burden, we postulate that a skewed T-cell cytokine response leads to plasma leakage in DHF. A better understanding of the immune responses associated with increased or decreased risk for DHF will be of immense value for the clinical studies of candidate multivalent DENV vaccines anticipated to take place in the next several years.

Intracellular cytokine production by dengue virus-specific T cells correlates with subclinical secondary infection.

The pathophysiology of dengue virus infection remains poorly understood, although secondary infection is strongly associated with more severe disease. In the present study, we performed a nested, case-control study comparing the responses of pre-illness peripheral blood mononuclear cells between children who would subsequently develop either subclinical or symptomatic secondary infection 6-11 months after the baseline blood samples were obtained and frozen. We analyzed intracellular cytokine production by CD4(+) and CD8(+) cells in response to stimulation with dengue antigen. We found higher frequencies of dengue virus-specific TNFα, IFNγ-, and IL-2-producing T cells among schoolchildren who subsequently developed subclinical infection, compared with those who developed symptomatic secondary dengue virus infection. Although other studies have correlated immune responses during secondary infection with severity of disease, to our knowledge this is the first study to demonstrate a pre-infection dengue-specific immune response that correlates specifically with a subclinical secondary infection.

B-cell responses during primary and secondary dengue virus infections in humans.

Low-avidity serotype-cross-reactive antibodies are hypothesized to play a key role in triggering severe disease in patients with secondary dengue virus (DENV) infection. However, there is little systematic information about the frequency, avidity, and cross-reactivity of DENV-specific B cells in individuals experiencing primary instead of secondary infection. We compared DENV-specific B-cell responses in a cohort of Thai children with primary or secondary DENV infection. B cells specific for DENV precursor membrane protein, envelope (E) protein, and nonstructural protein 1 were detectable in immune peripheral blood mononuclear cells with the highest frequencies of DENV E-specific B cells detected in patients experiencing primary DENV-1 infections. DENV E-specific B cells were highly serotype-specific after primary DENV infections, whereas most E-specific B cells in patients with secondary infection were serotype-cross-reactive and secreted antibodies with higher avidity to heterologous DENV serotypes. Our data suggest that the minor populations of serotype-cross-reactive B cells generated by primary DENV infection are preferentially expanded during secondary DENV infection.

Annotation of glycolysis, gluconeogenesis, and trehaloneogenesis pathways provide insight into carbohydrate metabolism in the Asian citrus psyllid.

Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid, Diaphorina citri. No curative treatment or significant prevention mechanism exists for this disease, which causes economic losses from reduced citrus production. A high-quality genome of D. citri is being manually annotated to provide accurate gene models to identify novel control targets and increase understanding of this pest. Here, we annotated 25 D. citri genes involved in glycolysis and gluconeogenesis, and seven in trehaloneogenesis. Comparative analysis showed that glycolysis genes in D. citri are highly conserved but copy numbers vary. Analysis of expression levels revealed upregulation of several enzymes in the glycolysis pathway in the thorax, consistent with the primary use of glucose by thoracic flight muscles. Manually annotating these core metabolic pathways provides accurate genomic foundation for developing gene-targeting therapeutics to control D. citri.

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-1ß>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-1ß 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.

Longitudinal Analysis of Dengue Virus-Specific Memory T Cell Responses and Their Association With Clinical Outcome in Subsequent DENV Infection.

Memory T cells resulting from primary dengue virus (DENV) infection are hypothesized to influence the clinical outcome of subsequent DENV infection. However, the few studies involving prospectively collected blood samples have found weak and inconsistent associations with outcome and variable temporal trends in DENV-specific memory T cell responses between subjects. This study used both ex-vivo and cultured ELISPOT assays to further evaluate the associations between DENV serotype-cross-reactive memory T cells and severity of secondary infection. Using ex-vivo ELISPOT assays, frequencies of memory T cells secreting IFN-γ in response to DENV structural and non-structural peptide pools were low in PBMC from multiple time points prior to symptomatic secondary DENV infection and showed a variable response to infection. There were no differences in responses between subjects who were not hospitalized (NH, n=6) and those who were hospitalized with dengue hemorrhagic fever (hDHF, n=4). In contrast, responses in cultured ELISPOT assays were more reliably detectable prior to secondary infection and showed more consistent increases after infection. Responses in cultured ELISPOT assays were higher in individuals with hDHF (n=8) compared to NH (n=9) individuals before the secondary infection, with no difference between these groups after infection. These data demonstrate an association of pre-existing DENV-specific memory responses with the severity of illness in subsequent DENV infection, and suggest that frequencies of DENV-reactive T cells measured after short-term culture may be of particular importance for assessing the risk for more severe dengue disease.

T lymphocyte responses to flaviviruses - diverse cell populations affect tendency toward protection and disease.

Dengue virus (DENV), Yellow Fever virus, West Nile virus, Japanese encephalitis virus and Zika virus are medically important flaviviruses transmitted to humans by mosquitoes and circulate in overlapping geographic areas. Cross-reactive immune responses have been demonstrated among the flaviviruses, particularly the four DENV serotypes. The immunological imprint left by a flavivirus infection can therefore have profound effects on the responses to subsequent infections. In this review we summarize recent research focusing on T cell responses to DENV using clinical samples from prospective cohort studies in Asia. These data suggest that durability of different T cell populations after natural infection or vaccination is an important consideration for the outcome of subsequent flavivirus exposures and we argue for continued investigation in the context of longitudinal cohort studies.

Vaccine innovations for emerging infectious diseases-a symposium report.

Vaccines have been incredibly successful at stemming the morbidity and mortality of infectious diseases worldwide. However, there are still no effective vaccines for many serious and potentially preventable infectious diseases. Advances in vaccine technology, including new delivery methods and adjuvants, as well as progress in systems biology and an increased understanding of the human immune system, hold the potential to address these issues. In addition, maternal immunization has opened an avenue to address infectious diseases in neonates and very young infants. This report summarizes the presentations from a 1-day symposium at the New York Academy of Sciences entitled "Innovative Vaccines against Resistant Infectious Diseases and Emerging Threats," held on May 20, 2019.

Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection.

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.