Novel adhesions, including trimeric autotransporters, might contribute to virulence.

BackgroundThe development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection.Methodology/Principal FindingsIn this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV), suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN) response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal) vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30.Conclusions/SignificanceThese results suggest that early transcriptional responses may be predictive of development of adaptive immunity to TDV vaccination in cynomolgus macaques, and will inform studies of human responses to dengue vaccines.

NCT00831012.

Word count (not including title, abstract, research in context, acknowledgment, references, tables, and figure legends): 5,077 RESEARCH IN CONTEXTEvidence before this study: Our PubMed search for articles matching the terms ("metagenomic" OR "cell-free DNA") and "infect*" in the title/abstract and using the "Human" species filter from inception to September 30, 2019 yielded 463 articles. Many proof-of-concept and validation studies illustrating how metagenomic sequencing can diagnose infections have been previously reviewed. Our search identified only nine studies which applied metagenomic shotgun sequencing to blood specimens, likely because there is a relatively low signal-to-noise ratio with this specimen type in this setting. In a study of 358 febrile sepsis patients, plasma cell-free DNA sequencing detected causative agents missed by standard-of-care testing in 15% of patients, but also detected bacterial organisms adjudicated as commensals in 10% of patients.Recently, a proof-of-concept study used machine learning to integrate metagenomic sequencing and transcriptional host response profiling to differentiate pathogens from commensal organisms in respiratory specimens, albeit with only a small derivation cohort to train host response signatures.Added value of this study: Our 200-patient study assessed the clinical utility of combining both metagenomic sequencing and a previously-defined host response assay to diagnose sepsis. We developed a rigorous chart review approach to measure whether our assays' results could change a physician's diagnostic decision-making, without having to commit the assays into patient care. Metagenomic sequencing revealed previously-undetected and clinically relevant organisms in 17 of 200 patients, and host response profiling led at least two of three physician chart reviewers to change SUMMARY Background: Current diagnostic techniques are inadequate for rapid microbial diagnosis and optimal management of patients with suspected sepsis. We assessed the impact of metagenomic sequencing and host response profiling individually and in combination on microbiological diagnosis in these patients. Methods:In this cohort study of 200 consecutive patients with suspected sepsis we evaluated three molecular diagnostic methods with blood specimens: 1) direct bacterial DNA detection and characterization with metagenomic shotgun next generation sequencing and contaminant sequence removal using Bayesian inference; 2) direct viral DNA and RNA enrichment and detection with viral capture sequencing; and 3) transcript-based host response profiling with a previously-defined 18-gene qRT-PCR assay. We then evaluated changes in diagnostic decision-making among three expert physicians in a chart review by unblinding our three molecular test results in a staged fashion.Findings: Metagenomic shotgun sequencing confirmed positive blood culture results in 14 of 26 patients. In 17 of 200 patients, metagenomic sequencing and viral capture sequencing revealed organisms that were 1) not detected by conventional...

199 words 29 Text: 3501 words (not including Acknowledgements) 30 31 Summary: Interferon-and cell cycle-associated gene transcript abundance levels in the 32 peripheral blood of dengue vaccine recipients on days 8 and 9 post-vaccination were associated 33 with dengue neutralizing antibody titers on day 42, and mirrored responses in primary dengue 34 infection, suggesting the possibility of predicting protective immunity. 35 36 3 ABSTRACT 37 38 Background: Several promising live attenuated virus (LAV) dengue vaccines are in 39 development, but information about innate immune responses and early correlates of protection 40 are lacking.41 Methods: We characterized human genome-wide transcripts in whole blood from 10 volunteers 42 at 11 time-points after immunization with the dengue virus type 3 (DENV-3) component of the 43 NIH dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary 44 DENV-3 infection. We compared day-specific gene expression patterns with subsequent 45 neutralizing antibody (NAb) titers. 46Results: The transcriptional response to vaccination was largely confined to days 5-20 and was 47 dominated by an interferon-associated signature and a cell cycle signature that peaked on days 48 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and 49 scope in symptomatic natural infection than following vaccination (maximum fold-change >200 50 versus 21 post-vaccination; 3,210 versus 286 transcripts with significant fold-change), but 51 shared gene modules were induced in the same sequence. The abundance of 131 transcripts 52 on days 8 and 9 post-vaccination was strongly correlated with NAb titers measured 6 weeks 53 post-vaccination.54 Conclusions: LAV dengue vaccination elicits early transcriptional responses that mirror those 55 found in symptomatic natural infection and provide candidate early markers of protection against 56 DENV infection.57 58 Clinical Trial Registration Number: NCT00831012 (available at clinicaltrials.gov) 59 60 of protection; interferon; neutralizing antibody 61 62 4 BACKGROUND 63 Each year, the four dengue virus serotypes (DENV-1-4) infect an estimated 390 million 64 individuals globally [1]. While most of these infections are asymptomatic, approximately 100 65 million individuals develop clinically apparent disease, from uncomplicated fever to life-66 threatening illness. Despite the high disease burden, there are no licensed therapeutics for 67 DENV infection. Several promising candidate dengue vaccines are in Phase III clinical trials, 68 and the live attenuated chimeric dengue vaccine Dengvaxia™ was recently licensed for use in 69 children 9 years of age and older in DENV endemic areas. However, the efficacy and duration 70 of protection were limited or uncertain, and DENV-naïve vaccine recipients were hospitalized for 71 dengue and severe dengue at a higher rate than placebo recipients, possibly due to antibody-72 dependent enhancement (ADE) [2]. 73 Studies of natural DENV infection and flavivirus LAVs have identifi...