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Responses by peripheral blood leukocytes may contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We used DNA microarrays to reveal transcriptional patterns in the blood of 14 adults with DHF. Acute DHF was defined by an abundance of transcripts from cell cycle— and endoplasmic reticulum (ER)—related genes, suggesting a proliferative response accompanied by ER stress. Transcript-abundance levels for immunoresponse-associated genes, including cell surface markers, immunoglobulin, and innate response elements, were also elevated. Twenty-four genes were identified for which transcript abundance distinguished patients with dengue shock syndrome (DSS) from those without DSS. All the gene transcripts associated with DSS, many of which are induced by type I interferons, were less abundant in patients with DSS than in those without DSS. To our knowledge, these data provide the first snapshot of gene-expression patterns in peripheral blood during acute dengue and suggest that DSS is associated with attenuation of selected aspects of the innate host response.

BACKGROUND: Several promising live attenuated dengue vaccines are in development, but information about innate immune responses and early correlates of protection is lacking. METHODS: We characterized human genome-wide transcripts in whole blood from 10 volunteers at 11 time points after immunization with the dengue virus type 3 (DENV-3) component of the National Institutes of Health dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary DENV-3 infection. We compared day-specific gene expression patterns with subsequent neutralizing antibody (NAb) titers. RESULTS: The transcriptional response to vaccination was largely confined to days 5–20 and was dominated by an interferon-associated signature and a cell cycle signature that peaked on days 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and scope in symptomatic natural infection than following vaccination (maximum fold-change >200 vs 21 postvaccination; 3210 vs 286 transcripts with significant fold-change), but shared gene modules were induced in the same sequence. The abundances of 131 transcripts on days 8 and 9 postvaccination were strongly correlated with NAb titers measured 6 weeks postvaccination. CONCLUSIONS: Live attenuated dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection. CLINICAL TRIALS REGISTRATION: NCT00831012.

Leptospirosis causes significant morbidity and mortality worldwide; however, the role of the host immune response in disease progression and high case fatality (>10–50%) is poorly understood. We conducted a multi-parameter investigation of patients with acute leptospirosis to identify mechanisms associated with case fatality. Whole blood transcriptional profiling of 16 hospitalized Brazilian patients with acute leptospirosis (13 survivors, 3 deceased) revealed fatal cases had lower expression of the antimicrobial peptide, cathelicidin, and chemokines, but more abundant pro-inflammatory cytokine receptors. In contrast, survivors generated strong adaptive immune signatures, including transcripts relevant to antigen presentation and immunoglobulin production. In an independent cohort (23 survivors, 22 deceased), fatal cases had higher bacterial loads (P = 0.0004) and lower anti-Leptospira antibody titers (P = 0.02) at the time of hospitalization, independent of the duration of illness. Low serum cathelicidin and RANTES levels during acute illness were independent risk factors for higher bacterial loads (P = 0.005) and death (P = 0.04), respectively. To investigate the mechanism of cathelicidin in patients surviving acute disease, we administered LL-37, the active peptide of cathelicidin, in a hamster model of lethal leptospirosis and found it significantly decreased bacterial loads and increased survival. Our findings indicate that the host immune response plays a central role in severe leptospirosis disease progression. While drawn from a limited study size, significant conclusions include that poor clinical outcomes are associated with high systemic bacterial loads, and a decreased antibody response. Furthermore, our data identified a key role for the antimicrobial peptide, cathelicidin, in mounting an effective bactericidal response against the pathogen, which represents a valuable new therapeutic approach for leptospirosis., Author Summary Leptospirosis causes over one million cases and nearly 60,000 deaths annually. Infection with the spirochetal bacterium results in a spectrum of symptoms, ranging from mild febrile illness to life-threatening pulmonary hemorrhage syndrome and acute kidney injury. Despite leptospirosis being a leading cause of zoonotic morbidity worldwide, little is known about the human immune response to Leptospira infections, and less about the pathogenic mechanisms resulting in severe disease outcomes. Here, we used a systems biology approach to discover transcripts and immunoprofiles associated with case fatality. We identified new risk factors for high bacterial loads and fatal leptospirosis, including the antimicrobial peptide, cathelicidin, which we validated in an animal model. Cathelicidin therefore represents a potential novel treatment for severe cases of leptospirosis.

Background The 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 Findings In 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/Significance These 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., Author Summary Dengue has become the leading cause of mosquito-borne virus infections worldwide. Despite considerable effort, development of a successful vaccine against dengue virus (DENV) has been challenging due to the co-circulation of the four DENV serotypes in endemic areas—to which humans develop distinct immune responses, and the increased risk of severe disease in those with pre-existing immunity to one serotype when they are infected with a different serotype. In this study, we investigated the responses in macaques to vaccination with the tetravalent, live-attenuated vaccine, TDV, by different doses and routes of vaccine administration. We identify changes in macaque gene expression that occurred in the days immediately following vaccination with TDV, a time-period that is difficult to study during natural infection. The gene expression response was characterized by features of the innate immune response to virus, notably the type I interferon response, and began the day after TDV vaccination. This response correlated with the development of neutralizing antibodies, which means that it might serve as an early indicator of a subsequent protective immune response to dengue vaccines.

DNA microarray-based gene transcript-profiling of the responses of primates to infection has begun to yield new insights into host-pathogen interactions; this approach, however, remains plagued by challenges and complexities that have yet to be adequately addressed. The rapidly changing nature over time of acute infectious diseases in a host, and the genetic diversity of microbial pathogens present unique problems for the design and interpretation of functional-genomic studies in this field. In addition, there are the more common problems related to heterogeneity within clinical samples, the complex, non-standardized confounding variables associated with human subjects and the complexities posed by the analysis and validation of highly parallel data. Whereas various approaches have been developed to address each of these issues, there are significant limitations that remain to be overcome. The resolution of these problems should lead to a better understanding of the dialogue between the host and pathogen.

Background To guide treatment of infectious diseases, clinicians need sensitive, specific, and rapid diagnostics. We aim to incorporate complementary methods of microbial sequencing and host-response profiling to improve the diagnosis of patients at risk for acute infections. Methods We enrolled 200 adult patients with systemic inflammatory response syndrome (SIRS) at the Stanford Emergency Department. Physicians with specialty training in infectious diseases conducted retrospective two-physician chart review to establish likely admission diagnoses. Blood samples were tested with a previously described 18-gene host-response integrated antibiotics decision model (IADM) that distinguishes noninfectious SIRS, bacterial infections and viral infections. Plasma samples were tested with shotgun metagenomic next-generation sequencing (NGS) and viral sequencing with VirCapSeq. A novel statistical algorithm was developed to identify contaminant organism sequences in NGS data. Results The physician chart review classified 99 patients (49%) as infected, 69 (35%) possibly infected and 32 (16%) non-infected. Compared with chart review, the IADM distinguished bacterial from viral infections with an area under curve of 0.85 (95% confidence interval 0.77–0.93). NGS results to date confirmed positive blood cultures in seven of nine patients, with two of four blood culture-positive E. coli patients turning up negative on NGS due to E. coli contamination. NGS also confirmed positive cultures from other sites in two of six patients with negative blood cultures. Preliminary VirCapSeq data from 23 patients confirmed positive viral tests in five of six patients with Hepatitis C, BK Virus, Cytomegalovirus and Epstein–Barr Virus infections. VirCapSeq did not identify a causative agent in the plasma of 11 patients with confirmed respiratory viral infection and intestinal Norovirus infection, and six patients with idiopathic illness. Interestingly, VirCapSeq found viral reactivation in 8 of 12 immunocompromised patients. Conclusion The diagnosis of suspected infections may be enhanced by integrating host-response and microbial data alongside clinical judgment. Our results and large cohort lay the foundation to demonstrate the utility of this approach and in which patients these tools may be most useful. Disclosures T. E. Sweeney, Inflammatix, Inc: Employee and Shareholder, Salary; T. Briese, Roche: Columbia University has licensed VirCapSeq to Roche, Licensing agreement or royalty; W. I. Lipkin, Roche: Columbia University has licensed VirCapSeq to Roche., Licensing agreement or royalty; P. Khatri, Inflammatix, Inc.: Co-founder, Scientific Advisor and Shareholder, Licensing agreement or royalty and ownership stock; D. A. Relman, Karius: Consultant, Stock options; Arc Bio LLC: Consultant, Stock options

Evaluation of immune mechanisms responsible for control of viral replication is critical to understanding HIV-2 attenuated biological characteristics in pathogenesis and transmission. Evaluation of the cellular immune response is often based on labor-intensive techniques that limit the scope of most studies performed. A simple and rapid anthrax toxin-based ELISPOT method to assess HIV-2 cellular immune response was developed. The modified anthrax toxin-based antigen presentation process performed better than a recombinant vaccinia system and the ELISPOT method significantly enhanced the ease and simplicity of the assay. Using this method, a robust HIV-2 cellular immune response directed toward the p26 core protein was exhibited in 21 of 24 (87.5%) infected women, and all 8 seronegative subjects were negative in both assays. Cellular immune responses were associated with low HIV-2 viral load. This simple and rapid modified anthrax toxin-based ELISPOT method allowed us to demonstrate, strong cellular immune responses that may be critical determinants in the HIV-2 attenuated phenotype.

A longitudinal cohort study was conducted to define the prevalence and temporal pattern of antibody response to the HIV-2 virion-associated proteins p26gag and Vpx. One hundred and forty-one asymptomatic HIV-2-infected women were enrolled, and followed for up to 11 years. Eighty-one percent of the subjects had antibodies to p26, and 51% to Vpx; response to these two antigens was not correlated. The response to both proteins was determined early in infection, and remained stable over time. The absence of antibodies to p26 was a highly significant predictor of CDC category IV HIV-related disease (p0.01) in both univariate and multivariate analysis. Antibody response to Vpx alone was not associated with disease progression. However, those individuals lacking anti-p26 antibodies, and with anti-Vpx antibodies, were six times more likely to be classified as CDC category IV by the end of the study (p0.01). This represents the first identification of virus-specific serological markers for HIV-2-related disease progression.

Efforts to discern the etiology of acute febrile disease are hampered by the paucity of reliable discriminating clinical features, difficulties in obtaining appropriate specimens, insensitive methods for detecting known causative agents, and the lack of diagnostic tests for some conditions associated with fever, such as autoimmune diseases and adverse drug reactions. As a result, many acute febrile illnesses remain unexplained, especially in the early days after onset of clinical signs and symptoms. Molecular profiling of the host response offers an approach for classifying acutely ill hosts that complements traditional diagnostic approaches based on microbial detection [1]. Studies of human genome–wide transcript abundance patterns in peripheral blood suggest that these patterns might provide useful information about the disease mechanism, outcome, nature of the infectious agent, and diagnosis [2–6]. This last possibility has not been adequately explored, especially in the setting of a clinical syndrome that presents important diagnostic dilemmas. Kawasaki disease (KD) is an acute, self-limited inflammatory illness of infants and children [7]; ~25% of untreated patients develop coronary artery aneurysms or ectasia. Intravenous immunoglobulin (IVIG) reduces the rate of coronary artery aneurysms to ~5% when administered within the first 10 days of illness, but KD remains the leading cause of acquired pediatric heart disease in developed nations. Despite 30 years of research, no etiologic agent has been identified for KD. In the absence of a specific diagnostic test, KD is diagnosed according to clinical criteria, many of which are shared by other illnesses characterized by rash or fever, including adenovirus infections, streptococcal scarlet fever, and systemic drug reactions. Many children with KD consequently receive an erroneous or late diagnosis, which leads to delays in treatment and an increased risk of coronary artery aneurysm formation [8, 9]. We recently examined whole-blood genomewide transcript abundance patterns in patients with KD and identified specific transcript levels associated with the risk of subsequent failure to respond to IVIG therapy [4]. In the current study, we compared patterns of whole-blood gene expression in patients during the acute phase of KD with patterns found in patients during the early phase of 3 illnesses that have similar clinical presentations but well-defined alternative etiologies. We identified patterns of gene expression and corresponding biological programs that were different in patients with KD, compared to patients with the other illnesses, and we were able to distinguish between KD and adenovirus infection on the basis of gene expression patterns. The results from this study indicate that comparative analysis of host gene expression profiles is a promising approach for better understanding febrile illnesses and that such analysis may contribute to the development of a test for KD that enables more accurate and timely diagnosis.

This paper demonstrates how the polymerase chain reaction can be used to increase the sensitivity of detection of Leishmania parasites by DNA hybridization methods through the amplification of the minicircle target sequence. The oligonucleotide primers used are able to direct the amplification of all Leishmania strains tested. In addition, the PCR products from L. mexicana and L. braziliensis strains can be distinguished by hybridization with kDNA probes. The method is sensitive enough to detect the kDNA from a single organism and this sensitivity allows the use of nonradioactive hybridization methods. This method can be used to detect Leishmania from human biopsy material.

The Tat protein of human immunodeficiency virus (HIV) is essential for viral replication and has extracellular pathogenic activity. We sought to determine whether the anti-Tat antibody response was predictive of disease progression in 144 HIV type 2 (HIV-2)-infected subjects observed longitudinally between 1985 and 2003. Sixty-eight percent of the subjects tested positive for anti-Tat antibodies, with reactivity notably established early after seroconversion and stably maintained over the course of infection. The risk and rate of progression to advanced HIV-2 AIDS was significantly higher in anti-Tat-negative subjects than in anti-Tat-positive subjects, extending the importance of this prognostic marker for HIV-2 AIDS.

Malaria is a global problem, and there is a critical need for further understanding of the disease process. When malarial parasites invade and develop within the bloodstream, they stimulate a profound host response whose main clinical sign is fever. To explore this response, we measured host gene expression in whole blood from Kenyan children hospitalized with either acute malaria or other febrile illnesses. Genomewide analysis of expression identified 2 principal gene-expression profiles related to neutrophil and erythroid activity. In addition to these general acute responses, a third gene-expression profile was associated with host parasitemia; mediators of erythrophagocytosis and cellular stress were notable components of this response. The delineation of subjects on the basis of patterns of gene expression provides a molecular perspective of the host response to malaria and further functional insight into the underlying processes of pathogenesis.

Human immunodeficiency virus type 2 (HIV-2), the second human retrovirus known to cause AIDS, is endemic to West Africa but is infrequently found outside this region. We present a case series of 10 HIV-2--infected individuals treated in the United States. Physicians applied the principles of highly active antiretroviral therapy (HAART), normally used in treating HIV type 1, with modifications considered appropriate for treating HIV-2. CD4+ cell count, HIV-2 virus load, and clinical status were found to correlate well, providing evidence that HIV-2 virus load is useful in managing treatment of patients with HIV-2 who are receiving therapy. However, HAART regimens with predicted efficacy for treatment of HIV type 1 infection are not as efficacious for treatment of HIV-2. Controlled clinical trials of HIV-2-infected patients receiving various HAART regimens are needed to provide therapeutic guidance to the medical community.

Human immunodeficiency virus type 2 (HIV-2) is less pathogenic than HIV type 1 (HIV-1), but the mechanisms underlying this difference have not been defined. We developed an internally controlled quantitative reverse transcriptase-polymerase chain reaction to measure HIV-2 viral load and determined levels of plasma virus in a cohort of registered commercial sex workers in Dakar, Senegal. The assay has a lower limit of detection of 100 copies/mL and is linear over 4 logs. HIV-2 viral RNA was detectable in 56% of all samples tested; the median load was 141 copies/mL. Levels of viral RNA in the plasma were inversely related to CD4+ cell counts. HIV-2 and HIV-1 viral loads were compared among the seroincident women in the cohort; the median viral load was 30x lower in the HIV-2-infected women (P

Dengue is the most prevalent mosquito-borne human illness worldwide. The ability to predict disease severity during the earliest days of the illness is a long-sought, but unachieved goal. We examined human genome-wide transcript abundance patterns in daily peripheral blood mononuclear cell (PBMC) samples from 41 children hospitalized with dengue virus (DENV) infection in Nicaragua, as well as 8 healthy control subjects. Nine patients had primary dengue fever (DF1), 11 had dengue fever with serologic evidence of prior DENV infection, i.e., secondary dengue fever (DF2), 12 had dengue hemorrhagic fever (DHF), and 9 had dengue shock syndrome (DSS). We identified 2,092 genes for which transcript abundance differed significantly between patients on days 3–6 of fever and healthy subjects (FDR, Author Summary Infection with dengue virus (DENV) causes dengue fever, the most prevalent mosquito-borne illness of humans worldwide. Tens of millions of cases occur annually; up to 500,000 patients develop additional life-threatening complications, including hemorrhage and shock. The clinical course of the disease evolves rapidly, making it difficult to identify patients at risk for severe disease and suggesting that biological events associated with the development of severe disease may be short-lived. We examined gene expression patterns in the blood of children hospitalized with DENV infection, and found that patients with differences in disease severity and history of previous DENV infection shared a common set of gene expression features, but the timing and magnitude of these features differed. In our study, prior DENV infection explained the greatest amount of variation in gene expression among patients. We discovered features of gene expression on day 3 that were associated with subsequent disease severity—features that were not apparent on subsequent days, emphasizing the importance of looking at temporal patterns of gene expression in acute infection.