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 gamma-chain knockout (NOD-scid IL2rgamma(null)) 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 IL2rgamma(null) mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha 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

A survey of health professions students for knowledge, attitudes, and confidence about tuberculosis, 2005

<p>Abstract</p> <p>Background</p> <p>In 2003 the NIH perceived a need to strengthen teaching about tuberculosis (TB) to health professions students. The National Tuberculosis Curriculum Consortium (NTCC) was funded to meet this need. The purpose of this study was to survey students enrolled in NTCC schools prior to NTCC-developed educational materials being made available to faculty.</p> <p>Methods</p> <p>A self-administered survey for students in NTCC schools to establish a baseline level of knowledge, attitudes, and confidence about tuberculosis.</p> <p>Results</p> <p>1480/2965 (50%) students in 28 programs in 20 NTCC schools completed the survey. If public health students are eliminated from totals (only 61 respondents of 765 public health students), the overall response proportion for the seven clinically-related disciplines was 64.5%. The majority (74%) were in schools of medicine (MD/DO), undergraduate nursing (BSN), and pharmacy (PharmD); others were in programs for physician assistants (PA), advanced practice nursing (NP/APN), respiratory therapy (RT), clinical laboratory sciences (MT/CLS), and public health (MPH). Almost 90% had attended at least one lecture about TB. Although 91.4% knew TB was transmitted via aerosols, about one-third did not know the method for administering tuberculin, or that Bacillus Calmette-Guerin (BCG) vaccine was not a contraindication to TB skin testing. Fewer than two-thirds knew that about 10% of people in the U.S.A. who have latent tuberculosis infection (LTBI) and a normal immune system will develop TB disease, or that BCG is not part of the routine vaccination program in the U.S.A. because it complicates surveillance for new TB infection.</p> <p>Conclusion</p> <p>There is room for improvement in knowledge, attitudes, and confidence about TB by health professions students surveyed. The NTCC-developed educational products may be used by faculty to improve student performance to be assessed with future surveys.</p

Decreased Dengue Replication and an Increased Anti-viral Humoral Response with the use of Combined Toll-Like Receptor 3 and 7/8 Agonists in Macaques

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo

Dengue Virus Infection-Enhancing Activity in Serum Samples with Neutralizing Activity as Determined by Using FcγR-Expressing Cells

Dengue has become a major international public health concern in recent decades. There are four dengue virus serotypes. Recovery from infection with one serotype confers life-long protection to the homologous serotype but only partial protection to subsequent infection with other serotypes. Secondary infection with a serotype different from that in primary infection increases the risk of development of severe complications. Antibodies may play two competing roles during infection: virus neutralization that leads to protection and recovery, or infection-enhancement that may cause severe complications. Progress in vaccine development has been hampered by limited understanding on protective immunity against dengue virus infection. We report the neutralization activity and infection-enhancement activity in individuals with dengue in Malaysia. We show that infection-enhancement activity is present when neutralizing activity is absent or low, and cross-reactive neutralizing activity may be hampered by infection-enhancing activity. Conventional assays for titration of neutralizing antibody do not consider infection-enhancement activity. We used an alternative assay that determines the sum of neutralizing and infection-enhancement activity in sera from dengue patients. In addition to providing insights into antibody responses during infection, the alternative assay provides a new platform for the study of immune responses to vaccine

Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

ABSTRACT: Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. METHODS: The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using N(G)-methyl L-Arginine (N(G)MLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. RESULTS: INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with N(G)MLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. CONCLUSION: This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production

Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. © 2013 Nascimento et al

Inhibition of Dengue Virus Entry and Multiplication into Monocytes Using RNA Interference

Prevention and treatment of dengue infection remain a serious global public health priority. Extensive efforts are required toward the development of vaccines and discovery of potential therapeutic compounds against the dengue viruses. Dengue virus entry is a critical step for virus reproduction and establishes the infection. Hence, the blockade of dengue virus entry into the host cell is an interesting antiviral strategy as it represents a barrier to suppress the onset of infection. This study was achieved by using RNA interference to silence the cellular receptor, and the clathrin mediated endocytosis that enhances the entry of dengue virus in monocytes. Results showed a marked reduction of infected monocytes by flow cytometry. In addition, both intracellular and extracellular viral RNA load was shown to be reduced in treated monocytes when compared to untreated monocytes. Based on these findings, this study concludes that this therapeutic strategy of blocking the virus replication at the first stage of multiplication might serve as a hopeful drug to mitigate the dengue symptoms, and reduction the disease severity

Pediatric Measles Vaccine Expressing a Dengue Antigen Induces Durable Serotype-specific Neutralizing Antibodies to Dengue Virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles–dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist