Showing papers by "Eva Harris published in 2010"

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Abstract: Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

Abstract: We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2.

Abstract: Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C' loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.

Trends in patterns of dengue transmission over 4 years in a pediatric cohort study in Nicaragua.

Abstract: Background Dengue is the most prevalent mosquito-borne viral disease in humans and a major urban public health problem worldwide.

Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis.

Abstract: Background Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

Abstract: Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5 untranslated region (5 UTR). Bootstrap analysis indicates that the 5 UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109. The genus Enterovirus (EV) in the family Picornaviridae is a group of related viruses that are associated with a spectrum of

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Abstract: Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.

High Dengue Case Capture Rate in Four Years of a Cohort Study in Nicaragua Compared to National Surveillance Data

Abstract: Dengue is a major public health problem in tropical and subtropical regions; however, under-reporting of cases to national surveillance systems hinders accurate knowledge of disease burden and costs. Laboratory-confirmed dengue cases identified through the Nicaraguan Pediatric Dengue Cohort Study (PDCS) were compared to those reported from other health facilities in Managua to the National Epidemiologic Surveillance (NES) program of the Nicaraguan Ministry of Health. Compared to reporting among similar pediatric populations in Managua, the PDCS identified 14 to 28 (average 21.3) times more dengue cases each year per 100,000 persons than were reported to the NES. Applying these annual expansion factors to national-level data, we estimate that the incidence of confirmed pediatric dengue throughout Nicaragua ranged from 300 to 1000 cases per 100,000 persons. We have estimated a much higher incidence of dengue than reported by the Ministry of Health. A country-specific expansion factor for dengue that allows for a more accurate estimate of incidence may aid governments and other institutions calculating disease burden, costs, resource needs for prevention and treatment, and the economic benefits of drug and vaccine development.

Clinical Attack Rate and Presentation of Pandemic H1N1 Influenza versus Seasonal Influenza A and B in a Pediatric Cohort in Nicaragua

Abstract: Background. Little is known about the clinical presentation and epidemiology of influenza A H1N1pdm in children in developing countries. We assessed the severity of influenza A H1N1pdm in children in Nicaragua by comparing H1N1pdm cases to seasonal influenza cases in an ongoing cohort study. Methods. The Nicaraguan Influenza Cohort Study was established in June 2007 to study the burden and seasonality of pediatric influenza in a tropical developing country. During the period from June 2007 through November 2009, a total of 4391 children aged 2‐14 years participated in the cohort. We examined the attack rate of clinical influenza and assessed symptoms at first presentation in febrile patients with H1N1pdm versus those with seasonal influenza A or B. Results. The estimated clinical attack rate of H1N1pdm in the cohort was 20.1%, compared to 11.7% and 15.1% for seasonal influenza A and 11.9% and 24.2% for seasonal influenza A and B in 2007 and 2008, respectively. Symptoms significantly associated with H1N1pdm cases versus seasonal influenza A cases were sore throat (adjusted odds ratio [OR], 1.7; 95% confidence interval [CI], 1.2‐2.5), wheezing (OR, 5.1; 95% CI, 1.3‐19.0), rhonchi (OR, 4.6; 95% CI, 1.4‐15.0), crepitations (OR, 16.2; 95% CI, 2.1‐128.7), pneumonia (OR, 8.0; 95% CI, 1.7‐37.3), nausea (OR, 2.8; 95% CI, 1.5‐5.1), and loss of appetite (OR, 2.1; 95% CI, 1.4‐3.1). In addition, 3 concurrent influenza and dengue virus coinfections were identified.

Gene Expression Patterns of Dengue Virus-Infected Children from Nicaragua Reveal a Distinct Signature of Increased Metabolism

Abstract: Author(s): Loke, P'ng; Hammond, Samantha N; Leung, Jacqueline M; Kim, Charles C; Batra, Sajeev; Rocha, Crisanta; Balmaseda, Angel; Harris, Eva | Abstract: BackgroundInfection with dengue viruses (DENV) leads to a spectrum of disease outcomes. The pathophysiology of severe versus non-severe manifestations of DENV infection may be driven by host responses, which could be reflected in the transcriptional profiles of peripheral blood immune cells.Methodology/principal findingsWe conducted genome-wide microarray analysis of whole blood RNA from 34 DENV-infected children in Nicaragua collected on days 3-6 of illness, with different disease manifestations. Gene expression analysis identified genes that are differentially regulated between clinical subgroups. The most striking transcriptional differences were observed between dengue patients with and without shock, especially in the expression of mitochondrial ribosomal proteins associated with protein biosynthesis. In the dengue hemorrhagic fever patients, one subset of differentially expressed genes encode neutrophil-derived anti-microbial peptides associated with innate immunity. By performing a meta-analysis of our dataset in conjunction with previously published datasets, we confirmed that DENV infection in vivo is associated with large changes to protein and nucleic acid metabolism. Additionally, whereas in vitro infection leads to an increased interferon signature, this was not consistently observed from in vivo patient samples, suggesting that the interferon response in vivo is relatively transient and was no longer observed by days 3-6 of illness.Conclusions/significanceThese data highlight important differences between different manifestations of severity during DENV infection as well as identify some commonalities. Compilation of larger datasets in the future across multiple studies, as we have initiated in this report, may well lead to better prediction of disease manifestation via a systems biology approach.

Index Cluster Study of Dengue Virus Infection in Nicaragua

Abstract: Traditional study designs do not identify acute asymptomatic or pre-symptomatic dengue virus (DENV) infections, thus limiting our understanding of immunologic and viral factors that modulate infection outcome. In the 2006 and 2007 dengue seasons, we conducted a pilot index cluster study in Managua, Nicaragua, in which 442 persons living within 50 meters of 22 index cases identified through an ongoing pediatric cohort study were evaluated for DENV infection. Post-enrollment and pre-enrollment DENV infections were confirmed in 12 (2.7%) and 19 (4.3%) contacts, respectively. Five (42%) post-enrollment infections were asymptomatic, and DENV-2 was identified in 9 (75%) infections. Phylogenetic analysis with full-length DENV genomic sequence from contacts, index cases, and cohort dengue cases indicated focal transmission and infection outside the local area. We demonstrate the feasibility of identification of acute asymptomatic and pre-symptomatic cases in urban Latin America, the first report of such a study in the Americas, and identify age and concomitant immunity to DENV of contacts as a key factor in index cluster study design.

Diagnostic accuracy of a rapid influenza test for pandemic influenza A H1N1

Abstract: With the current influenza A H1N1 pandemic (H1N1pdm), it is extremely important that clinicians can quickly and accurately identify influenza cases Methodology/Principal Findings: To investigate the performance of the QuickVue Influenza A+B rapid test, we conducted a prospective study of the diagnostic accuracy of the QuickVue Influenza A+B test compared to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for influenza A H1N1pdm in Nicaraguan children aged 2 to 14 years Rapid test sensitivity and specificity compared to real-time RT-PCR were 641% (95% CI 535, 739) and 983% (950, 996), respectively Agreement between the two tests was 864% (95% CI 817, 903), and kappa was calculated to be 067 (95% CI 056, 076) Performance of the rapid test varied by day of presentation, with a sensitivity of 417% (95% CI 221, 634) for samples from children presenting on the day of symptom onset and a sensitivity of 721% (95% CI 599, 823) for samples from children presenting one or more days post-symptom onset Conclusions/Significance: We found that the rapid test performed with moderate sensitivity and high specificity Test performance varied by day of onset, with lower sensitivity on the day of symptom onset

On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis

Abstract: We have demonstrated a postprocessed complementary metal oxide semiconductor (CMOS) integrated circuit (IC) capable of on-chip magnetic separation, i.e., removing via magnetic forces the nonspecifically bound magnetic beads from the detection area on the surface of the chip. Initially, 4.5 μm wide superparamagnetic beads sedimenting out of solution due to gravity were attracted to the detection area by a magnetic concentration force generated by flowing current through a conductor embedded in the IC. After sedimentation, the magnetic beads that did not bind strongly to the functionalized surface of the IC through a specific biochemical complex were removed by a magnetic separation force generated by flowing current through another conductor placed laterally to the detection area. As the spherical bead pivoted on the surface of the chip, the lateral magnetic force was further amplified by mechanical leveraging, and 50 mA of current flowing through the separation conductor placed 18 μm away from the bead re...

Severe coinfections of dengue and pandemic influenza A H1N1 viruses.

Abstract: Here we report on 4 hospitalized patients with dengue-influenza virus coinfections. All patients were RT-PCR positive for dengue virus and pandemic influenza A H1N1. Clinical findings at presentation ranged from influenza-like illness to severe dengue. Clinical progression of the infections varied, but all developed dengue symptoms and had interstitial infiltrates. Three cases required intensive care management and 1 case was fatal.

Sensitive population profiling and genome assembly of HIV and Flaviviruses using ultra-deep sequencing technologies

Abstract: Viral diseases such as HIV/AIDS and Dengue have an enormous impact on human health worldwide. Despite this, application of new sequencing technologies to viral genomics has lagged. We are using genome sequence data to study how populations of single stranded RNA viruses, including HIV, Dengue, West Nile and Hepatitis C, evolve within infected individuals in response to host immune, therapeutic and vaccine pressures. To support this, we have developed high-throughput sequencing, assembly and population profiling pipelines based on 454 and Illumina technology that are tuned to the specific needs of viral sequencing. These strategies can capture full genome sequences and can profile sequence diversity at each residue in the genome with unprecedented sensitivity. Our analytical pipeline for 454 and Illumina data (i) generates complete genome assemblies from short sequencing reads derived from populations with high rates of variation; and (ii) detects and quantifies variants in these populations with high sensitivity, while differentiating true variants from process errors. Initial results with our viral sequencing and analysis pipeline are extremely promising. We detected rare variants to below 1% frequency, revolutionizing our ability to accurately assess the earliest events in viral evolution. We have demonstrated effective assessment of genome-wide diversity during acute HIV infection, enabling rapid, affordable, and highly sensitive identification of the earliest cellular immune responses to HIV. This has allowed us to detect earlier evolutionary events, demonstrating, for example, that HIV cytotoxic T-lymphocyte (CTL) escape can occur much faster than previously known. In addition, we have shown that the extent of intra-host diversity in Flaviviruses such as DENV and WNV is different between these closely related viruses with the latter exhibiting greater genetic diversity. The results presented here demonstrate the power of scalable, next generation sequencing-based methodologies as a genome-wide and unbiased global approach to profiling genomic diversity in intra-host populations of single stranded RNA viruses.

NIAID Workshop on Flavivirus Immunity

Abstract: On September 16, 2009, the National Institute of Allergy and Infectious Diseases (NIAID), part of the U.S. National Institutes of Health, convened a workshop to discuss current knowledge of T- and B-cell immune epitopes for members of the Flavivirus genus (family Flaviviridae), and how this information could be used to increase our basic understanding of host-pathogen interactions and/or advance the development of new or improved vaccines and diagnostics for these pathogens. B-cell and T-cell responses to flaviviruses are critical components of protective immunity against these pathogens. However, they have also been linked to disease pathogenesis. A detailed understanding of the biological significance of immune epitope information may provide clues regarding the mechanisms governing the induction of protective versus pathogenic adaptive immune responses.