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Background Three-dose series of conventional alum-adjuvanted Hepatitis B surface antigen (HBsAg)-based vaccines achieve seroprotection rates (SPRs) of 35-70% in PLWH. HepB-CpG, a HBsAg vaccine adjuvanted with a TLR-9 agonist, achieves high SPR in immunocompetent adults with a 2-dose regimen, but limited data exist in PLWH. Methods A5379 is an ongoing prospective, open-label study to evaluate immunogenicity of the HBV vaccine HepB-CpG in PLWH. The HBV vaccine-naïve group consisted of PLWH without past HBV infection on antiretroviral therapy with CD4 ≥100 cells/mm3 and HIV-1 RNA < 1000 copies/mL. Participants received 0.5 mL of HepB-CpG intramuscularly (20 mcg recombinant HBsAg and 3000 mcg of CpG 1018® adjuvant) at Wks 0, 4, and 24. Primary objectives were to determine the proportion of participants who achieve seroprotection (HBsAb≥10 mIU/mL) at Wk 28 and to assess safety. This study was designed to conclude SPR >55%, with up to 10% loss to follow-up prior to 28 wks. Results Of the 74 eligible participants enrolled at 13 global sites: 46% were male, 66% Asian, 16% Black,15% White and median age was 47 years (range 23-68). 27% were enrolled in the US, 65% in Thailand. Median CD4 was 625 cells/mm3, 96% had HIV-1 RNA < 60 copies/mL, and 9% had diabetes. Primary analysis set per analysis plan consisted of 68 participants (excluded: 3 missed visit, 3 out of visit window). All 68 completed 3 doses and achieved seroprotection (100% [95% CI: 94.7%, 100%]). 88% had HBsAb >1000 mIU/mL (assay upper limit). The SPR was also 100% [CI: 94.2%,100%] in the per protocol analysis set of 62 participants. At 8 wks after the 2nd dose, the SPR was 94.4% followed by 98.5% at Wk 24, prior to the 3rd dose. The proportion of participants with HBsAb >1000 mIU/mL increased from 27.9% at Wk 24 to 83.8% at Wk 28. One or more AEs related to study treatment were experienced by 61% of participants (39% Grade 1, 20% Grade 2, Grade 3 malaise in one participant). Vaccination site pain (40%), malaise (26%), fatigue (23%), myalgia (22%) and headache (22%) were the most frequent AEs. Seroprotection achieved by study week. Percentage of participants achieving seroprotection over the first 28 study weeks. HepB-CpG vaccine was administered at Entry, Week 4, and Week 24. Conclusion In this study of PLWH with no history of HBV vaccination or evidence of prior HBV exposure, 100% seroprotection was achieved at 4 weeks after 3 doses of the HepB-CpG. No unexpected safety issues were observed. Disclosures Kristen Marks, MD, MS, Gilead Sciences: Grant/Research Support Andrea L. Cox, M.D. Ph.D., Janssen: Advisor/Consultant Jennifer C. Price, MD, PhD, AbbVie: Grant/Research Support|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Merck: Grant/Research Support|Theratechnologies: Advisor/Consultant Kelvin McKoy, M.D., M.B.A., Dynavax Technologies: Employee|Dynavax Technologies: Ownership Interest|Dynavax Technologies: Stocks/Bonds Kenneth E. Sherman, MD, PhD, AbbVie: Grant/Research Support|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Helio: Grant/Research Support|Inovio: DSMB|Intercept: Grant/Research Support|MedPace: DSMB|Theratechnologies: Advisor/Consultant|Zydus: Grant/Research Support.

ObjectiveThe rectal microbiome was examined to assess the relationship between the microbiome and liver disease in HIV-infection.DesignEighty-two HIV-1 mono-infected individuals from the PROSPEC-HIV-study (NCT02542020) were grouped into three liver health categories based on results of controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) of transient elastography: normal (n = 30), steatosis (n = 30), or fibrosis (n = 22).MethodsLiver steatosis and fibrosis were defined by CAP at least 248 dB/m and LSM at least 8.0 kPa, respectively. 16S rRNA gene and whole genome shotgun metagenomic sequencing were performed on rectal swabs. Bacterial differences were assessed using zero-inflated negative binomial regression and random forests modeling; taxonomic drivers of functional shifts were identified using FishTaco.ResultsLiver health status explained four percentage of the overall variation (r2 = 0.04, P = 0.003) in bacterial composition. Participants with steatosis had depletions of Akkermansia muciniphila and Bacteroides dorei and enrichment of Prevotella copri, Finegoldia magna, and Ruminococcus bromii. Participants with fibrosis had depletions of Bacteroides stercoris and Parabacteroides distasonis and enrichment of Sneathia sanguinegens. In steatosis, functional analysis revealed increases in primary and secondary bile acid synthesis encoded by increased Eubacterium rectale, F. magna, and Faecalibacterium prausnitzii and decreased A. muciniphila, Bacteroides fragilis and B. dorei. Decreased folate biosynthesis was driven by similar changes in microbial composition.ConclusionHIV mono-infection with steatosis or fibrosis had distinct microbial profiles. Some taxa are similar to those associated with non-alcoholic fatty liver disease in HIV-negative populations. Further studies are needed to define the role of the gut microbiota in the pathogenesis of liver disease in HIV-infected persons.

BackgroundAlterations in the gut microbiome have been associated with HIV infection, but the relative impact of HIV versus other factors on the gut microbiome has been difficult to determine in cross-sectional studies.MethodsTo address this, we examined the gut microbiome, serum metabolome, and cytokines longitudinally within 27 individuals before and during acute HIV using samples collected from several ongoing cohort studies. Matched control participants (n=28) from the same cohort studies without HIV but at similar behavioral risk were used for comparison.FindingsWe identified few changes in the microbiome during acute HIV infection, but did find alterations in serum metabolites involving secondary bile acid (lithocholate sulfate, glycocholenate sulfate) and amino acid metabolism (3-methyl-2-oxovalerate, serine, cysteine, N-acetylputrescine). Greater microbiome differences, including decreased Bacteroides spp and increased Megasphaera elsdenii, were seen when comparing pre-HIV infection visits to matched at-risk controls. Those who acquired HIV also had elevated inflammatory cytokines (TNF-α, B cell activating factor, IL-8) and bioactive lipids (palmitoyl-sphingosine-phosphoethanolamide and glycerophosphoinositol) prior to HIV acquisition compared to matched controls.InterpretationLongitudinal sampling identified pre-existing microbiome differences in participants with acute HIV compared to matched control participants observed over the same period. These data highlight the importance of increasing understanding of the role of the microbiome in HIV susceptibility.FundingThis work was supported by NIH/NIAID (K08AI124979; P30AI117943), NIH/NIDA (U01DA036267; U01DA036939; U01DA036926; U24DA044554), and NIH/NIMH (P30MH058107; R34MH105272).

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in western countries both in children and adults. Metabolic dysregulation associated with gut microbial dysbiosis may influence disease progression from hepatic steatosis to inflammation and subsequent fibrosis. Using a multi-omics approach, we profiled the oral and fecal microbiome and plasma metabolites from 241 predominantly Latino children with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), and controls. Children with more severe liver pathology were dysbiotic and had increased gene content associated with lipopolysaccharide biosynthesis and lipid, amino acid and carbohydrate metabolism. These changes were driven by increases in Bacteroides and concomitant decreases of Akkermansia, Anaerococcus, Corynebacterium, and Finegoldia. Non-targeted mass spectrometry revealed perturbations in one-carbon metabolism, mitochondrial dysfunction, and increased oxidative stress in children with steatohepatitis and fibrosis. Random forests modeling of plasma metabolites was highly predictive of non-alcoholic steatohepatitis (NASH) (97% accuracy) and hepatic fibrosis, steatosis and lobular inflammation (93.8% accuracy), and can differentiate steatohepatitis from simple steatosis (90.0% accuracy). Multi-omics predictive models for disease and histology findings revealed perturbations in one-carbon metabolism, mitochondrial dysfunction, and increased oxidative stress in children with steatohepatitis and fibrosis. These results highlight the promise of non-invasive biomarkers for the growing epidemic of fatty liver disease.

Combinations of antibiotics have shown promise in off-label treatment of Inflammatory Bowel Disease (IBD) flares in adult and pediatric patients1–6. In a small pediatric cohort of moderate-severe medically refractory Ulcerative Colitis (UC), half achieved remission with a regimen of 3–4 antibiotics6. The rationale is to decrease mucosal inflammation by altering intestinal microbiota, however, to date no study has documented these changes. We report our experience with combination oral antibiotics in symptomatic pediatric patients with steroid-dependent or -resistant UC, and demonstrate significant alterations in gut microbiota during and after antibiotic therapy with reductions in inflammatory markers.

Background Oral daily tenofovir (TFV) disoproxil fumarate/emtricitabine (TDF/FTC) for human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) is highly effective for HIVprevention, yet long-term effects are not fully understood. We investigated the effects of PrEP on the rectal microbiome in a cohort of men who have sex with men (MSM). Methods This cross-sectional analysis included HIV-negative MSM either on PrEP (n = 37) or not (n = 37) selected from an ongoing cohort using propensity score matching. Rectal swabs were used to examine microbiome composition using 16S ribosomal ribonucleic acid gene sequencing, and associations between PrEP use and microbiota abundance were examined. Hair specimens were used to quantify TFV and FTC exposure over the past 6 weeks on a subset of participants (n = 15). Results Pre-exposure prophylaxis use was associated with a significant increase in Streptococcus abundance (adjusted P = .015). Similar associations were identified using least absolute shrinkage and selection operator (LASSO) regression, confirming the increase in Streptococcus and also showing increased Mitsuokella, Fusobacterium, and decreased Escherichia/Shigella. Increased Fusobacterium was significantly associated with increasing TFV exposure. Conclusions Oral TDF/FTC for PrEP is associated with rectal microbiome changes compared to well matched controls, specifically increased Streptococcus and Fusobacterium abundance. This study highlights the need for future investigations of the role of microbiome changes on HIV susceptibility and effectiveness of PrEP.

Background Recent advances in sequencing technologies and bioinformatics tools have allowed for large-scale microbiome studies that are rapidly advancing medical research. However, small changes in technique or analysis can significantly alter the results and lead to conflicting findings. Quantifying the technical versus biological variation expected in targeted 16S rRNA gene sequencing studies and how this variation changes with input biomass is critical to guide meaningful interpretation of the current literature and plan future research. Results Data were compiled from 469 sequencing libraries across 19 separate targeted 16S rRNA gene sequencing runs over a 2.5-year time period. Following removal of contaminant sequences identified from negative controls, 244 samples retained sufficient reads for further analysis. Coefficients of variation for intra- and inter-assay variation from repeated measurements of a bacterial mock community ranged from 8.7 to 37.6% (intra) and 15.6 to 80.5% (inter) for all but one genus of bacteria whose relative abundance was greater than 1%. Intra- versus inter-assay Bray-Curtis pairwise distances for a single stool sample were 0.11 versus 0.31, whereas intra-assay variation from repeat stool samples from the same donor was greater at 0.38 (Wilcoxon p = 0.001). A dilution series of the bacterial mock community was used to assess the effect of input biomass on variability. Pairwise distances increased with more dilute samples, and estimates of relative abundance became unreliable below approximately 100 copies of the 16S rRNA gene per microliter. Using this data, we created a prediction model to estimate the expected variation in microbiome measurements for given input biomass and relative abundance values. Conclusions Well-controlled microbiome studies are sufficiently robust to capture small biological effects and can achieve levels of variability consistent with clinical assays. Relative abundance is negatively associated with measures of variability and has a stronger effect on variability than does absolute biomass, suggesting that it is feasible to detect differences in bacterial populations in very low-biomass samples. Further, by quantifying the effect of biomass and relative abundance on compositional variability, we developed a tool for defining the expected variance in a given microbiome study. Electronic supplementary material The online version of this article (10.1186/s40168-018-0543-z) contains supplementary material, which is available to authorized users.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

Metabolic dysregulation associated with gut microbial dysbiosis can influence the progression of nonalcoholic fatty liver disease (NAFLD) from hepatic steatosis to inflammation and subsequent fibrosis. Using a multi-omics approach, we identified microbiome-derived biochemicals and genes associated with progressive hepatic pathology and fibrosis in Latino children (N=241). Children with more severe liver pathology were dysbiotic and had increased gene content associated with lipopolysaccharide biosynthesis and lipid, amino acid and carbohydrate metabolism. These changes were driven by increases in Bacteroides and concomitant decreases of Akkermansia, Anaerococcus, Corynebacterium, and Finegoldia. Nontargeted mass spectrometry revealed perturbations in one-carbon metabolism, mitochondrial dysfunction, and increased oxidative stress in children with steatohepatitis and fibrosis. Random forests modeling of plasma metabolites were highly predictive of nonalcoholic steatohepatitis (NASH) (97% accuracy) and hepatic fibrosis, steatosis and lobular inflammation (93.8% accuracy), and can differentiate steatohepatitis from simple steatosis (90.0% accuracy). These metabolomic biomarkers of hepatic pathology provide insights for therapeutic targeting of bacterially-mediated and host pathways.

Establishment of the infant microbiome has lifelong implications on health and immunity. Gut microbiota of breastfed compared with nonbreastfed individuals differ during infancy as well as into adulthood. Breast milk contains a diverse population of bacteria, but little is known about the vertical transfer of bacteria from mother to infant by breastfeeding.To determine the association between the maternal breast milk and areolar skin and infant gut bacterial communities.In a prospective, longitudinal study, bacterial composition was identified with sequencing of the 16S ribosomal RNA gene in breast milk, areolar skin, and infant stool samples of 107 healthy mother-infant pairs. The study was conducted in Los Angeles, California, and St Petersburg, Florida, between January 1, 2010, and February 28, 2015.Amount and duration of daily breastfeeding and timing of solid food introduction.Bacterial composition in maternal breast milk, areolar skin, and infant stool by sequencing of the 16S ribosomal RNA gene.In the 107 healthy mother and infant pairs (median age at the time of specimen collection, 40 days; range, 1-331 days), 52 (43.0%) of the infants were male. Bacterial communities were distinct in milk, areolar skin, and stool, differing in both composition and diversity. The infant gut microbial communities were more closely related to an infant's mother's milk and skin compared with a random mother (mean difference in Bray-Curtis distances, 0.012 and 0.014, respectively; P .001 for both). Source tracking analysis was used to estimate the contribution of the breast milk and areolar skin microbiomes to the infant gut microbiome. During the first 30 days of life, infants who breastfed to obtain 75% or more of their daily milk intake received a mean (SD) of 27.7% (15.2%) of the bacteria from breast milk and 10.3% (6.0%) from areolar skin. Bacterial diversity (Faith phylogenetic diversity, P = .003) and composition changes were associated with the proportion of daily breast milk intake in a dose-dependent manner, even after the introduction of solid foods.The results of this study indicate that bacteria in mother's breast milk seed the infant gut, underscoring the importance of breastfeeding in the development of the infant gut microbiome.

Targeting of the PI3K (phosphoinositide3-kinase)/Akt/mTOR pathway in human ovarian cancer cells is a promising novel therapeutic strategy. We investigated the effects of cisplatin and the PI3K inhibitor LY294002 on invasion, migration and the expression of essential matrix metalloproteinases (MMPs) in ovarian cancer cells. SKOV3, OVCAR5 and IGROV1 human ovarian cancer cell lines were treated with cisplatin, LY294002 and a combination of both drugs. Invasion and migration of treated cells was assessed using Matrigel and uncoated PET membrane assays. Expression levels of pro-MMP2, MMP2, TIMP1, TIMP2 and MT1-MMP were determined using Western Blotting. Gel zymography was used to quantitate the functional levels of active MMP2. All three cell lines showed significantly reduced invasion and migration after treatment with cisplatin, LY294002, and the combination of both drugs compared to untreated controls. In SKOV3 cells, cisplatin alone and in combination with LY294002 resulted in a 6.3 and 7.1-fold reduction in the total amount of activated MMP2. TIMP1 expression decreased by 5.0, 6.6 and 28.4-fold with cisplatin, LY294002 and the combination respectively (P < 0.05). In contrast, only cisplatin and the combination of both drugs resulted in a significant, 3.7 and 5.1-fold reduction in the level of TIMP2. Expression levels of MT1-MMP remained unchanged. These observations were corroborated in IGROV1 cell lines that showed similar changes of activated MMP2 and TIMP2 expression, but no significant decrease in TIMP1 levels. Our data suggests that inhibition of ovarian cancer cell motility is mediated via down-regulation of activated MMP2, TIMP1 and TIMP2 expression under these treatment conditions.

Introduction: Ovarian cancer is the most lethal of the gynecological malignancies and the 5th leading cause of cancer related deaths amongst women in the US. It is a chemotherapy-sensitive disease, and about 85% of patients respond to first line treatment with platinum-based chemotherapeutic agents. However, 15% of all patients present with primary platinum-resistant disease, and those with recurrent ovarian cancer develop resistance. Resistance to platinum-based drugs is a major obstacle in ovarian cancer treatment, and mechanisms of platinum resistance are not well understood. Insulin-like growth factor-1 receptor (IGF-1R) signaling has not only been implicated in ovarian cancer development, but has also been suggested to mediate platinum-resistance. The IGF-1R has been proposed as a potential molecular target for ovarian cancer, and clinical trials evaluating the use of small molecule kinase inhibitors or humanized monoclonal antibodies against the IGF-1R are currently under way. This study examines the role of IGF-1R in cisplatin resistance in human ovarian carcinoma. Experimental Procedures: Differential gene expression in two pairs of cisplatin-resistant cell lines (SKOV3-CisR and OVCAR-CisR) and their syngeneic cisplatin-sensitive counterpart (SKOV3 and OVCAR5, respectively) was analyzed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. IGF-1R mRNA transcripts and protein levels were verified via qRT-PCR and immunoblotting, respectively. Protein expression levels of IGF-1Rα and IGF-1Rα (immunoblotting) were correlated with cisplatin IC50 (XTT cell viability) in a panel of 12 ovarian cancer cell lines. IGF-1R was overexpressed (stable transfection) in SKOV3-CisR cells and the effect on cisplatin sensitivity evaluated. The effect of short-term cisplatin treatment on IGF-1Rα and IGF-1Rα protein levels was studied in SKOV3 and OVCAR5 cells. Results: Cisplatin-resistant SKOV3-CisR and OVCAR5-CisR cells exhibited 2-3 fold lower levels of IGF-1R mRNA transcripts compared to cisplatin-sensitive SKOV3 and OVCAR5 cells in the DNA microarray and via qRT-PCR. Correspondingly, decreased levels of IGF-1Rα and IGF-1Rα protein were found in immunoblot analyses. IGF-1Rα (r = −0.6118; p = 0.0345) and IGF-1Rα (r = −0.5839; p = 0.0462) protein levels and cisplatin IC50were negatively correlated. IGF-1R overexpression in SKOV3-CisR cells sensitized the cells to cisplatin, from 5 μg/ml (SKOV3-CisR) to 10-12 μg/ml (SKOV3-CisR-IGF-1R). Cisplatin induced a decrease in IGF-1Rα and IGF-1Rα levels after 24 and 48 hours treatment. Conclusions: Cisplatin treatment of ovarian cancer cells results in IGF-1R downregulation, which in turn decreases cisplatin sensitivity. Our results have important implications in the design of IGF-1R targeting strategies in clinical trials for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 836. doi:1538-7445.AM2012-836