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ObjectiveWe aimed to evaluate the association between cumulative illness with neurodevelopment and growth outcomes in a birth cohort of Guatemalan infants.Study DesignFrom June 2017 to July 2018, infants 0-3 months of age living in a resource-limited region of rural southwest Guatemala were enrolled and completed weekly at-home surveillance for caregiver-reported cough, fever and vomiting/diarrhea. They also underwent anthropometric assessments and neurodevelopmental testing with the Mullen Scales of Early Learning (MSEL) at enrollment, six months, and one year.ResultsOut of 499 enrolled infants, 430 (86.2%) completed all study procedures and were included in the analysis. At 12-15 months of age, 140 (32.6%) infants had stunting (length-for-age Z [LAZ] score ConclusionsThese findings highlight the negative cumulative consequences of frequent febrile and respiratory illness on neurodevelopment during infancy. Future studies should explore the inflammatory profile associated with these syndromic illnesses and their impact on neurodevelopment in the first years of life.

Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. RNA-Seq technology has matured in recent years and is now widely deployed for transcriptional analysis of clinical specimens. The goal of this study was to benchmark technical parameters of RNA-Seq in the context of a multi-site, double-blind randomized clinical trial using primary patient samples. We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two large-scale replicate datasets. We evaluated the impact of: (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data, and established the impact of read depth and gene filtering on transcript representation. Lastly, we modeled statistical power to detect DE genes for a range of sample sizes, effect sizes, and coverage depths. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Although Central America is largely dengue virus (DENV)-endemic, the 2015–2016 Zika virus (ZIKV) pandemic brought new urgency to develop surveillance approaches capable of characterizing the rapidly changing disease burden in resource-limited settings. We conducted a pediatric DENV surveillance study in rural Guatemala, including serial cross-sectional surveys from April through September 2015 (Survey 1), in October–November 2015 (Survey 2), and January–February 2016 (Survey 3). Serum underwent DENV IgM MAC ELISA and polymerase chain reaction testing. Using banked specimens from Surveys 2 and 3, we expanded testing to include DENV 1–4 and ZIKV microneutralization (MN50), DENV NS1 IgG ELISA, and ZIKV anti-NS1 antibody Blockage of Binding (BoB) ELISA testing. Demographic risk factors for ZIKV BoB positivity were explored using multivariable generalized linear regression models. Of Survey 2 and 3 samples available (N = 382), DENV seroprevalence slightly increased (+1%–10% depending on the assay) during the surveillance period and increased with age. In contrast, ZIKV seroprevalence consistently increased over the 3-month period, including from 6% to 34% (P < 0.0001) and 10%–37% (P < 0.0001) using the MN50 ≥100 and BoB ELISA assays, respectively. Independent risk factors for ZIKV seropositivity included older age (prevalence ratio (PR)/year = 1.12, 95% confidence interval (CI) = 1.07–1.17) and primary caregiver literacy (PR = 2.80, CI = 1.30–6.06). Rapid active surveillance (RAS) surveys demonstrated a nearly 30% increase in ZIKV prevalence and a slight (≤ 10%) increase in DENV seroprevalence from October to November 2015 to January to February 2016 in rural southwest Guatemala, regardless of serologic assay used. RAS surveys may be a useful “off-the-shelf” tool to characterize arboviruses and other emerging pathogens rapidly in resource-limited settings.

Antibody dependent enhancement has been well described between Zika and dengue viruses, but is poorly characterized between West Nile and dengue viruses. We demonstrate that neuroinvasive West Nile virus infection leads to the development of non-neutralizing, cross-reactive IgG antibodies to dengue and Zika viruses capable of causing antibody dependent enhancement in vitro of dengue virus and leads to the formation of flavivirus cross-reactive memory B cells in some patients.

Background Genogroup II noroviruses are the most common cause of acute infectious gastroenteritis. We evaluated the use of a new GII.2 inoculum in a human challenge. Methods Forty-four healthy adults (36 secretor-positive and 8 secretor-negative for histo-blood group antigens) were challenged with ascending doses of a new safety-tested Snow Mountain virus (SMV) GII.2 norovirus inoculum (1.2 × 104 to 1.2 × 107 genome equivalent copies [GEC]; n = 38) or placebo (n = 6). Illness was defined as diarrhea and/or vomiting postchallenge in subjects with evidence of infection (defined as GII.2 norovirus RNA detection in stool and/or anti-SMV immunoglobulin G [IgG] seroconversion). Results The highest dose was associated with SMV infection in 90%, and illness in 70% of subjects with 10 of 12 secretor-positive (83%) and 4 of 8 secretor-negative (50%) becoming ill. There was no association between prechallenge anti-SMV serum IgG concentration, carbohydrate-binding blockade antibody, or salivary immunoglobulin A and infection. The median infectious dose (ID50) was 5.1 × 105 GEC. Conclusions High rates of infection and illness were observed in both secretor-positive and secretor-negative subjects in this challenge study. However, a high dose will be required to achieve the target of 75% illness to make this an efficient model for evaluating potential norovirus vaccines and therapeutics. Clinical Trials Registration NCT02473224.

In a phase 1 randomized, single-center clinical trial, inactivated influenza virus vaccine delivered through dissolvable microneedle patches (MNPs) was found to be safe and immunogenic. Here, we compare the humoral and cellular immunologic responses in a subset of participants receiving influenza vaccination by MNP to the intramuscular (IM) route of administration. We collected serum, plasma, and peripheral blood mononuclear cells in 22 participants up to 180 days post-vaccination. Hemagglutination inhibition (HAI) titers and antibody avidity were similar after MNP and IM vaccination, even though MNP vaccination used a lower antigen dose. MNPs generated higher neuraminidase inhibition (NAI) titers for all three influenza virus vaccine strains tested and triggered a larger percentage of circulating T follicular helper cells (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) compared to the IM route. Our study indicates that inactivated influenza virus vaccination by MNP produces humoral and cellular immune response that are similar or greater than IM vaccination.

Molecular detection of dengue virus (DENV) RNA from serum or plasma provides an accurate acute-phase diagnostic (< 7 days after symptom onset). Detection may be prolonged in whole blood, although data are limited. We tested for DENV by real-time reverse transcription–PCR in 345 paired acute-phase plasma and whole blood samples from individuals with a Flavivirus-like illness in southwestern Guatemala. In 18/18 cases with detectable DENV RNA in plasma, whole blood samples were positive and yielded similar cycle threshold values. In seven individuals with convalescent samples obtained 2–3 weeks later, DENV RNA remained detectable in whole blood but not plasma. In three additional cases, DENV RNA was only detectable in whole blood at the acute visit. In two cases, whole blood detection was linked to a virologically confirmed DENV infection 6–11 weeks earlier. Whole blood DENV RNA detection is sensitive for acute dengue infection and may remain positive for weeks to months.

Antibody cross-reactivity confounds testing for dengue virus (DENV) and Zika virus (ZIKV). We evaluated anti-DENV and anti-ZIKV IgG detection using a multiplex serological platform (the pGOLD assay, Nirmidas, Palo Alto, CA) in patients from the Asunción metropolitan area in Paraguay, which experiences annual DENV outbreaks but has reported few autochthonous ZIKV infections. Acute-phase sera were tested from 77 patients who presented with a suspected arboviral illness from January to May 2018. Samples were tested for DENV and ZIKV RNA by real-time reverse transcription-PCR, and for DENV nonstructural protein 1 with a lateral-flow immunochromatographic test. Forty-one patients (51.2%) had acute dengue; no acute ZIKV infections were detected. Sixty-five patients (84.4%) had anti–DENV-neutralizing antibodies by focus reduction neutralization testing (FRNT50). Qualitative detection with the pGOLD assay demonstrated good agreement with FRNT50 (kappa = 0.74), and quantitative results were highly correlated between methods (P < 0.001). Only three patients had anti–ZIKV-neutralizing antibodies at titers of 1:55–1:80, and all three had corresponding DENV-neutralizing titers > 1:4,000. Hospitalized dengue cases had significantly higher anti-DENV IgG levels (P < 0.001). Anti-DENV IgG results from the pGOLD assay correlate well with FRNT, and quantitative results may inform patient risk stratification.

(1) Background: The influenza virus continues to cause significant annual morbidity and mortality. The overall efficacy of seasonal influenza vaccination is suboptimal, which is partly due to host immune factors. The effects of imprinting and repeated seasonal influenza vaccination were investigated to assess for immune factors and mechanisms that impact influenza vaccine responses. (2) Methods: Twenty participants were enrolled into a prospective pilot study based on birth cohort and seasonal influenza immunization history. Immunologic parameters were assessed over a six-month period after the seasonal influenza vaccine was administered. (3) Results: There was no significant imprinting effect, as measured by hemagglutination inhibition (HAI) fold change, HAI geometric mean titer (GMT) for Day 29 or Day 180 post-vaccination and antigen- specific antibody-secreting cells (ASC) for Day 8 post-vaccination. Individuals who had minimal prior seasonal influenza vaccination had a higher magnitude ASC response and a higher HAI fold change post-vaccination than individuals who were repeatedly vaccinated. (4) Conclusions: Repeated seasonal influenza vaccination resulted in a decreased fold change of the immune response, although individuals in this cohort tended to have high HAI titers at baseline that persisted after vaccination. Imprinting effects were not observed in this cohort. These host immune factors should be considered in the development of universal influenza vaccines. ClinicalTrials.gov Identifier: NCT03686514

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barre syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.

Background Little is known about the epidemiology of Cytomegalovirus (CMV) infection in low resource countries. We evaluated the frequency and effects of post-natal CMV infection in infants from a prospective cohort study designed to assess the effects of post-natal Zika on neurodevelopment (ND) in rural Guatemala. Infants with CMV infection (blue bars) were older compared CMV-negative (red bars) infants. Methods Infants were evaluated for CMV infection by PCR using urine samples collected at 0-3 months of age. ND testing was conducted by local psychologists using a culturally adapted Mullen Scales of Early Learning (MSEL). We explored associations between CMV infection and microcephaly, neurological, visual and hearing deficits, malnutrition and ND outcomes at 1 year of age. Results The infant cohort (N = 469) had a mean age at enrollment of 1.5 (SD 0.75) months; 47% were female and 71% were breastfeeding at 1 year. A total of 103 (22%) were CMV positive and the majority of these (97%) were > 4 weeks of age at testing. Infants > 4 weeks of age were more likely to be CMV positive (P < 0.0001) (Figure). Gender was not correlated with CMV positivity. Among children with head circumference (HC) measurements, microcephaly (HC < 2 SD) was present in 9/87 (10.3%) CMV positive and 35/338 (10.4%) CMV negative infants at 0-3 months of age (p =0.99). Among 438 infants who underwent screening for hearing deficits and a complete ophthalmologic evaluation, none of the CMV positive children had abnormal vision or hearing. Abnormal neurological exams in the first year of life occurred in 50/100 (50%) CMV positive and 166/365 (45.5%) CMV negative infants (p =0.56). There was no association between CMV infection at 0-3 months and MSEL overall or subdomain scores at 1 year (overall Relative risk (RR) 1.02, 95% CI 0.99-1.05, p=0.16). Malnutrition at 0-3 months (RR: 1.53, 95% CI 0.89-2.66, p = 0.13) and 1 year (RR: 1.10, 95% CI 0.77-1.58, p=0.59) was not associated with CMV infection at 0-3 months. Conclusion In a cohort of Guatemalan infants, postnatal CMV infection was common (22%) and more likely to occur after the neonatal period. There was no correlation between CMV infection and microcephaly at 0-3 months or at 1 year of age, nor with abnormal nutritional, neurologic, ophthalmologic, hearing or ND deficits at 1 year of age. This is the first epidemiologic report on CMV infection in early life in rural Guatemala. Disclosures Molly Lamb, PhD, BioFire (Grant/Research Support)

Chikungunya virus (CHIKV) is an alphavirus that is primarily transmitted by Aedes species mosquitoes. Though reports of an illness consistent with chikungunya date back over 200 years, CHIKV only gained worldwide attention during a massive pandemic that began in East Africa in 2004. Chikungunya, the clinical illness caused by CHIKV, is characterized by a rapid onset of high fever and debilitating joint pain, though in practice, etiologic confirmation of CHIKV requires the availability and use of specific laboratory diagnostics. Similar to infections caused by other arboviruses, CHIKV infections are most commonly detected with a combination of molecular and serological methods, though cell culture and antigen detection are reported. This review provides an overview of available CHIKV diagnostics and highlights aspects of basic virology and epidemiology that pertain to viral detection. Although the number of chikungunya cases has decreased since 2014, CHIKV has become endemic in countries across the tropics and will continue to cause sporadic outbreaks in naive individuals. Consistent access to accurate diagnostics is needed to detect individual cases and initiate timely responses to new outbreaks.

Liquid-chromatography mass spectrometry is commonly used to identify and quantify metabolites from biological samples to gain insight into human physiology and pathology. Metabolites and their abundance in biological samples are labile and sensitive to variations in collection conditions, handling and processing. Variations in sample handling could influence metabolite levels in ways not related to biology, ultimately leading to the misinterpretation of results. For example, anticoagulants and preservatives modulate enzyme activity and metabolite oxidization. Temperature may alter both enzymatic and non-enzymatic chemistry. The potential for variation induced by collection conditions is particularly important when samples are collected in remote locations without immediate access to specimen processing. Data are needed regarding the variation introduced by clinical sample collection processes to avoid introducing artifact biases. In this study, we used metabolomics and lipidomics approaches paired with univariate and multivariate statistical analyses to assess the effects of anticoagulant, temperature, and time on healthy human plasma samples collected to provide guidelines on sample collection, handling, and processing for vaccinology. Principal component analyses demonstrated clustering by sample collection procedure and that anticoagulant type had the greatest effect on sample metabolite variation. Lipids such as glycerophospholipids, acylcarnitines, sphingolipids, diacylglycerols, triacylglycerols, and cholesteryl esters are significantly affected by anticoagulant type as are amino acids such as aspartate, histidine, and glutamine. Most plasma metabolites and lipids were unaffected by storage time and temperature. Based on this study, we recommend samples be collected using a single anticoagulant (preferably EDTA) with sample processing at &lt, 24 h at 4 &deg, C.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids, several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

The re-emergence of Zika virus (ZIKV) in the western hemisphere has most significantly affected dengue virus (DENV) endemic regions. Due to the geographical overlap between these two closely related flaviviruses, numerous individuals who suffered ZIKV infection during recent outbreaks may have also previously been exposed to DENV. As such, the impact of pre-existing dengue immunity on immune responses to ZIKV has been an area of focused research and interest. To understand how B cell responses to a ZIKV infection may be modulated by prior dengue exposures, we compared and contrasted plasmablast repertoire and specificity between two ZIKV-infected individuals, one dengue-na&iuml, ve (ZK018) and the other dengue-experienced (ZK016). In addition to examining serological responses, we generated 59 patient plasmablast-derived monoclonal antibodies (mAbs) to define the heterogeneity of the early B cell response to ZIKV. Both donors experienced robust ZIKV-induced plasmablast expansions early after infection, with comparable mutational frequencies in their antibody variable genes. However, notable differences were observed in plasmablast clonality and functional reactivity. Plasmablasts from the dengue-experienced donor ZK016 included cells with shared clonal origin, while ZK018 mAbs were entirely clonally unrelated. Both at the mAb and plasma level, ZK016 antibodies displayed extensive cross-reactivity to DENV1-4, and preferentially neutralized DENV compared to ZIKV. In contrast, the neutralization activity of ZK018 mAbs was primarily directed towards ZIKV, and fewer mAbs from this donor were cross-reactive, with the cross-reactive phenotype largely limited to fusion loop-specific mAbs. ZK016 antibodies caused greater enhancement of DENV2 infection of FcR&gamma, expressing cells overall compared to ZK018, with a striking difference at the plasma level. Taken together, these data strongly suggest that the breadth and protective capacity of the initial antibody responses after ZIKV infection may depend on the dengue immune status of the individual. These findings have implications for vaccine design, given the likelihood that future epidemics will involve both dengue-experienced and na&iuml, ve populations.

Background Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. Results We enrolled 45 cases and 14 controls. Commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%), and arthralgia (82.2% and 54.5%), respectively. The sensitivity (94%) and duration of infection detection (80% positivity at 65–79 days after disease onset) by polymerase chain reaction were highest in whole-blood specimens. ZIKV-neutralizing antibodies had a half-life of 105 days and were significantly higher in dengue virus–experienced cases than naïve ones (P = .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. Conclusions ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.

Objective The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed >40 years ago either are not dynamic or fail to capture low levels of central nervous system (CNS) inflammation. Therefore, we aimed to identify and validate biomarkers of CNS inflammation in 2 blinded, prospectively acquired cohorts of untreated patients with neuroimmunological diseases and embedded controls, with the ultimate goal of developing clinically useful tools. Methods Because biomarkers with maximum utility reflect immune phenotypes, we included an assessment of cell specificity in purified primary immune cells. Biomarkers were quantified by optimized electrochemiluminescent immunoassays. Results Among markers with cell-specific secretion, soluble CD27 is a validated biomarker of intrathecal T-cell activation, with an area under the receiver operating characteristic curve of 0.97. Comparing the quantities of cerebrospinal fluid (CSF) immune cells and their respective cell-specific soluble biomarkers (released by CSF cells as well as their counterparts in CNS tissue) provided invaluable information about stationary CNS immune responses, previously attainable via brain biopsy only. Unexpectedly, progressive and relapsing–remitting multiple sclerosis (MS) patients have comparable numbers of activated intrathecal T and B cells, which are preferentially embedded in CNS tissue in the former group. Interpretation The cell-specific biomarkers of intrathecal inflammation may improve diagnosis and management of neuroimmunological diseases and provide pharmacodynamic markers for future therapeutic developments in patients with intrathecal inflammation that is not captured by imaging, such as in progressive MS. Ann Neurol 2015;78:3–20

Understanding the immune response during acute Zika in humans will aid vaccine design and testing. In 5 acute patients, including 2 pregnant women, viral levels and innate, T-, and B-cell responses against Zika or dengue viruses are described., Background There is an urgent need for studies of viral persistence and immunity during human Zika infections to inform planning and conduct of vaccine clinical trials. Methods In 5 returned US travelers with acute symptomatic Zika infection, clinical features, viral RNA levels, and immune responses were characterized. Results Two pregnant, flavivirus-experienced patients had viral RNA persist in plasma for >44 and >26 days. Three days after symptom onset, transient increases in proinflammatory monocytes began followed at 5 days by transient decreases in myeloid dendritic cells. Anti-Zika virus immunoglobulin M was detected at day 7 after symptom onset, persisted beyond 103 days, and remained equivocal through day 172. Zika virus–specific plasmablasts and neutralizing antibodies developed quickly; dengue virus–specific plasmablasts and neutralizing antibodies at high titers developed only in flavivirus-experienced patients. Zika virus– and dengue virus–specific memory B cells developed in both flavivirus-naive and -experienced patients. CD4+ T cells were moderately activated and produced antiviral cytokines after stimulation with Zika virus C, prM, E, and NS5 peptides in 4/4 patients. In contrast, CD8+ T cells were massively activated, but virus-specific cells that produced cytokines were present in only 2/4 patients assessed. Conclusions Acute infections with Zika virus modulated antigen-presenting cell populations early. Flavivirus-experienced patients quickly recalled cross-reactive MBCs to secrete antibodies. Dengue virus–naive patients made little dengue-specific antibody but developed MBCs that cross-reacted against dengue virus. Zika virus–specific functional CD4+ T cells were readily detected, but few CD8+ T cells specific for the tested peptides were found.

Background The host immune response to influenza vaccination can be affected by prior imprinting to a specific influenza strain based on birth cohort and prior influenza vaccination history. Understanding the underlying immune mechanisms is essential to development of an improved seasonal vaccine and an effective universal influenza vaccine. Methods This is a prospective pilot study, with a total of 20 subjects in either the H3N2 cohort (N = 10, born 1968–1977) or the H1N1 cohort (N = 10, born 1948–1957). Each cohort was further stratified by subjects who have received the influenza vaccine < 2 or ≥ 3 of the past 5 years. The FDA-approved quadrivalent 2018–19 influenza vaccine (containing A(H1N1), an A/Michigan/45/2015-like virus; A(H3N2), an A/Singapore/INFIMH-16–0019/2016-like virus; B/Colorado/06/2017-like virus; and B/Phuket/3073/2013-like virus) was administered on Day 1. Demographic information included age, gender, ethnicity, and BMI. HAI titers for each component of the vaccine were obtained at baseline, 29 days post-vaccination, and 180 days post-vaccination. HAI fold-change and HAI geometric mean titers (GMT) were analyzed. Results There was no significant difference between H1N1 or H3N2 HAI fold-change in the H3N2 birth cohort (P = 0.7496) or in the H1N1 birth cohort (P = 0.8237), Figure A. Comparing HAI fold-change for the repeatedly vs. minimally vaccinated groups, there was a significant higher fold change in the minimally vaccinated group (H1N1 HAI (P = 0.002) and H3N2 HAI (P < 0.0001), Figure B). GMT was higher at baseline for the repeatedly vaccinated group (H1N1, 70; H3N2, 98; vs. H1N1, 30; H3N2, 21 for the minimally vaccinated group); however, the GMT for the minimally vaccinated group was higher at day 29 (H1N1, 172; H3N2, 184; vs. H1N1, 422; H3N2, 299 for the minimally vaccinated group; Figure C). HAI titers and analysis at day 180 post vaccination are in progress. Conclusion There was no evidence of an imprinting effect by birth cohort for HAI titer magnitudes, even when stratified by vaccination history. There was a significantly higher HAI fold change for individuals who had received minimal influenza vaccinations in the past 5 years at 29 days post-vaccination. Individuals who had repeated vaccinations in the last 5 years had higher HAI GMT at baseline. Disclosures Nadine Rouphael, MD, Merck: I conduct as Emory PI the PNEUMO MERCK study at Emory, Research Grant; Pfizer: I conduct as co-PI the RSV PFIZER study at Emory, Research Grant; Sanofi-Pasteur: I conducted as Emory PI the CDIFFENSE trial at Emory, Research Grant.

Despite the widespread outbreak, few cases of Zika virus associated with cardiac manifestations have been described. We present a case of pericarditis in the setting of an acute, symptomatic Zika virus infection in a traveler returning from St. Thomas. Clinicians should be alert for this potential complication of Zika virus infection.

During the current Zika virus (ZIKV) outbreak, acute symptomatic ZIKV infection in adults appears to be a mild-to-moderate, self-limited illness. We present a case of ZIKV rash illness that improved and then relapsed without repeat exposure to ZIKV. Clinicians should be alert for relapses in patients with ZIKV infection.

Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies.

Background The clinical, virologic and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US patients with suspected Zika virus (ZIKV) infection were enrolled and clinical data and specimens were prospectively collected. Body fluids were tested for ZIKV RNA by PCR and blood was tested using serologic and cellular immune assays. Findings from those with confirmed ZIKV infections (cases) and ZIKV-negative controls were compared. Results We enrolled 45 cases and 14 controls. The most commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%) and arthralgia (82.2% and 54.5%), respectively. The sensitivity and duration of detection by PCR were highest in whole blood samples (94% of 35 cases who had samples collected up to day 79 post illness onset were positive); strikingly, 84% of those were still positive at 65–79 days post illness onset (Figure 1). ZIKV neutralizing antibodies were detected in all cases and none of the controls, and titers were significantly higher in dengue virus (DENV)-experienced subjects than in DENV-naïve ones (Figure 2). Among cases, anti-ZIKV IgG antibodies were also significantly higher in DENV-experienced patients, while anti-ZIKV IgM antibodies were no higher in DENV-experienced compared with naïve ones. Using intracellular cytokine staining, the highest frequencies of T cells producing IFN-γ, IL-2 and/or TNF-α were against the NS1, NS3, and NS5 proteins for CD4+ T cells, and against the E, NS3, and NS5 proteins for CD8+ T cells (Figure 3). Conclusion Detection of ZIKV RNA was more frequent and much more prolonged in whole blood samples compared with other body fluids. Diagnostic molecular assays on this easily obtained fluid should be prioritized for point-of-care development. Robust cellular responses to E, NS3 and NS5 proteins could have implications for vaccine development. Disclosures R. L. Atmar, Takeda Vaccines, Inc.: Investigator, Research grant.

BACKGROUND: Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development. METHODS: KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers. RESULTS: The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5FI +) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5FI +) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5FI + cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5FI+ cells in culture. However, this resulted in decreased germ cell gene expression. CONCLUSIONS: From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.