Intestinal Dysbiosis Following Cholestasis is Reduced by Active Immunization with a Detoxified Endotoxin Vaccine

Research Article

J Bacteriol Mycol. 2015;2(1): 1011.

Intestinal Dysbiosis Following Cholestasis is Reduced by Active Immunization with a Detoxified Endotoxin Vaccine

Samuel M. Alaish¹*, Emmanuel F. Mongodin², Lei Zhang³, Ebony Murphy¹ and Alan Cross³

1Department of Surgery, University of Maryland School of Medicine, Baltimore

2Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore

3Center for Vaccine Development, University of Maryland School of Medicine, Baltimore

*Corresponding author: Samuel M. Alaish, M.D. Assistant Professor of Surgery, Department of Surgery, University of Maryland School of Medicine, 29 South Greene St, Suite 110 Baltimore, MD 21201

Received: January 22, 2015; Accepted: April 16, 2015; Published: April 17, 2015


Background: Cholestasis inevitably leads to intestinal barrier loss resulting in endotoxemia and sepsis. Using a cholestatic mouse model, we have previously reported decreased intestinal barrier resistance, increased rates of bacterial translocation, endotoxemia and increased mortality following cholestasis. Furthermore, we found an intestinal dysbiosis, an altered gut microbiota, with increased numbers of virulent bacterial species 2 weeks after cholestatic injury as compared to sham-operated mice. We hypothesize that active immunization with the detoxified endotoxin vaccine, J5dLPS, will prevent the dysbiosis seen following cholestasis.

Methods: C57Bl/6J mice were vaccinated with a detoxified endotoxin vaccine, J5dLPS. Saline-injected mice served as controls. Following confirmation of serum antibody titers, mice then underwent either a Common Bile Duct Ligation (CBDL) as a model for cholestatic injury or a sham operation. Both before surgery and one week following surgery, stool specimens were collected for bacterial DNA isolation and sequencing.

Results: In this pilot study, J5dLPS vaccination was highly immunogenic and well-tolerated in C57Bl/6J mice. No dysbiosis was seen following active immunization. Moreover, J5dLPS-vaccinated mice which underwent CBDL demonstrated reduced intestinal dysbiosis and a strong trend [p=0.07] toward no dysbiosis compared to the dysbiosis seen in saline-injected mice after CBDL.

Conclusions: The detoxified endotoxin vaccine, J5dLPS, does not cause an intestinal dysbiosis in mice which have been actively immunized. This finding provides additional evidence of safety supporting future clinical use of this vaccine. Moreover, this same vaccine greatly reduces the gut dysbiosis seen in mice following cholestatic injury. Future studies are warranted to determine if maintenance of the gut microbiota strengthens the intestinal barrier following cholestasis and prevents gut colonization with opportunistic pathogens.

Keywords: Cholestasis; Endotoxin; Vaccine; Microbiota


Infants can develop Short Bowel Syndrome (SBS) following an extensive bowel resection for either a congenital anomaly or a postnatal infection [1]. Consequently, they develop intestinal failure and require prolonged intravenous nutrition [1]. Moreover, these infants can experience diminished bile flow from the liver into the intestine, known as cholestasis. Cholestasis leads to liver damage and also to intestinal barrier breakdown with increased rates of systemic infection. The organisms often responsible for these infections arise from the gut and coincide with intraluminal bacterial overgrowth, which follows both cholestasis [2] and SBS [3]. Moreover, a vicious cycle is created as intestinal permeability increases with bacterial overgrowth [4].

Failure of the intestinal barrier has been shown to be a characteristic feature of cholestatic injury. Decreased intestinal resistance, increased bacterial translocation and increased episodes of sepsis have been well described [5-7]; however, the exact mechanisms remain poorly understood. Using Common Bile Duct Ligation (CBDL) in a mouse as a model for cholestatic intestinal injury and bacterial overgrowth, our lab has previously found that C57BL/6J mice developed an intestinal dysbiosis, an altered gut microbiota, as demonstrated on a Principal Coordinates Analysis 14 days following cholestasis; however, a different strain of inbred mice, the A/J strain, failed to develop a dysbiosis [8]. Importantly, the dysbiosis positively correlated with the decreased intestinal resistance, increased bacterial translocation and increased mortality observed in the cholestatic C57BL/6J mice as compared to the cholestatic A/J mice [8]. We found a relative increase in the number of both Clostridiae and Proteobacteriae organisms and a relative decrease in Lactobacillae in the cholestatic C57BL/6J mice compared to their non-cholestatic counterparts [8]. This may be indicative of a transition towards a more pathogenic microbiota following cholestasis which three weeks later contributes to the significant mortality difference between the strains. Furthermore, we found that soluble CD14 levels, a marker of the monocytic response to LPS, were dramatically increased in all mice fourteen days following CBDL compared to shams (p<0.000002), thus representative of LPS translocation from the gut into the circulation [8].

Translocation of gut bacteria and endotoxin into the systemic circulation may contribute to the morbidity and mortality of cholestasis; therefore, we immunized mice with a detoxified endotoxin vaccine that was previously shown to elicit antibodies against the core region of LPS that is highly conserved among Enterobacteriaceae. Antibodies generated against the LPS core are highly protective in animal models of sepsis when induced actively or administered passively [9-12]. This vaccine has been well-tolerated when given to human subjects [13].

We hypothesize that a vaccine directed against endotoxin will offset some of the morbidity and mortality associated with cholestasis. In this study, we examine the effects of the detoxified endotoxin vaccine, J5dLPS, on the intestinal microbiota before and after cholestasis. The findings from this study shed light on potential safety concerns regarding the vaccine as well as an additional, potential therapeutic indication.

Materials and Methods


The J5dLPS vaccine was developed as previously described [13].


Male C57BL/6J (B6) mice (8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogenfree animal facility with 12-hour light-dark cycles. All mice weighed 18 to 25 g at the time of operation. Animal studies were conducted according to protocols reviewed and approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee and adhered to guidelines promulgated by the National Institutes of Health.

Common Bile Duct Ligation (CBDL) model of cholestasis

Mice were anesthetized by inhaled isoflurane anesthesia. The abdomen was clipped and then prepared in sterile fashion with 70% ethyl-ethanol followed by betadine. A transverse upper abdominal incision was performed. The Common Bile Duct (CBD) was dissected away from the portal vein and was ligated near its junction with the duodenum using aneurysm clips engineered with a precisely standardized opening/closing mechanism. The abdominal wall was then closed in a two-layer fashion using absorbable sutures. Shamoperated mice were treated identically but without dissection or ligation of the CBD. Postoperatively, animals were resuscitated with warmed subcutaneous injections of saline (1 mL) to replace losses. Mice were returned to clean cages where food and water were provided ad libitum.

Vaccine schedule and measurement of antibody levels

The J5dLPS vaccine was administered with an intraperitoneal injection using a dose of 20μg (based on dLPS content) at 0, 2, and 4 weeks. Saline-injected mice were immunization controls. After an additional 4 weeks, the CBDL was performed. Sham-operated mice served as controls. Blood samples were collected 1 month after the final immunization (before the CBDL or sham operation). Levels of antibodies against the core glycolipid of LPS were measured using a standard ELISA method as previously described and expressed as Optical Density Units (ODUs) [13].

Gut Bacterial Community Profiling

DNA extraction: Profiling of the bacterial communities inhabiting the gut of male C57BL/6J (B6) mice was performed as earlier described [14]. Total genomic DNA was extracted from fecal pellets using the protocol previously described by Zupancic and colleagues [14]. Briefly, 1 ml of phosphate-buffered saline was added to the mouse stool aliquots, and cell lysis was performed using an enzymatic cocktail composed of lyzosyme, mutanolysin and lysostaphin. After 1-hour incubation at 37oC, samples were further lysed by addition of proteinase K and 10% SDS, followed by incubation at 55oC for 45 minutes. The samples were then disrupted by bead beating using a FP120 Fast Prep instrument and 0.1 mm silica spheres. The resulting crude lysate was processed using the ZYMO Fecal DNA Kit (Zymogen) according to the manufacturer–s recommendations. Negative extraction controls, where stool samples were omitted, were performed to ensure the samples were not contaminated by exogenous bacterial DNA during the extraction process.

16S rRNA gene sequencing: Following DNA extraction, the universal primers 27F and 338R were used for PCR amplification of the V1–V3 hypervariable regions of 16S rRNA genes. The 338R primer included a unique sequence tag to barcode each sample. Using 96 barcoded 338R primers, the V1–V3 regions of 16S rRNA genes were amplified in 96-well microtiter plates using AmpliTaq Gold DNA polymerase (Applied Biosystems) and 50 ng of template DNA in a total reaction volume of 50 mL, using the cycling conditions described by Zupancic and colleagues [14]. Negative controls without a template were included for each barcoded primer pair. PCR products were quantified using the Quant-iT PicoGreen dsDNA assay, and equimolar amounts (100 ng) of the PCR amplicons were mixed in a single tube. The purified amplicon mixture was then sequenced by 454 FLX Titanium pyrosequencing using 454 Life Sciences primer A by the Genomics Resource Center at the Institute for Genome Sciences, University of Maryland School of Medicine, using protocols recommended by the manufacturer as amended by the Center.

16S rRNA gene sequences statistical analysis: Processing (sequence binning and trimming) and analysis of the 16S rRNA reads was performed using the CLoVR system (CloVR 1.0-RC5; https:// and the CloVR-16S protocol ( clovr-16s; version 1.1), and the output of the CLoVR pipeline processed further using the Qiime software package, as well as the Phyloseq package implemented in R. Phylogenetic trees in Figures 2 & 3 were created using the Phyloseq plot tree function from the tree generated in Qiime using the command. The tree was constructed with a set of sequences representative of the OTUs, using the defaults Qiime parameters (Fast Tree for tree building). Sobs analysis of the number of Observed Operational Taxonomic Units (OTUs) was performed with p<0.05 considered significant. The Shannon Diversity index and the Chao1 richness estimate were calculated using Phyloseq to determine species diversity and richness, respectively.

Phylogenetic trees: The phylogenetic trees were created using the Phyloseq plot tree function, from the tree generated in Qiime using the command. The Qiime tree was constructed with a set of sequences representative of the OTUs aligned against a database of reference 16S sequences (in this case, Green genes), using the defaults Qiime parameters, i.e. using Fast Tree for tree building. Fast Tree infers approximately-maximumlikelihood phylogenetic trees from alignments of nucleotide sequences, and can handle alignments with up to a million of sequences in a reasonable amount of time and memory. Using the default parameters, Fast Tree estimates the reliability of each split in the tree by computing local support values with the Shimodaira- Hasegawa test, and does not use traditional bootstrapping: this is why there are no bootstrap values for these trees.

Statistical analysis

The experiment was replicated once. A paired Student–s t-test was used to measure antibody levels with p<0.05 considered significant. The Wilcoxon rank sum test was used to determine significance when comparing OTU–s, Chao1 richness estimates and Shannon diversity estimates. P<0.05 was considered significant.


Immunogenicity of J5dLPS vaccine

A strong antibody response was demonstrated in 7 of the 8 mice one month following 3 doses of 20μg of J5dLPS vaccine given 2 weeks apart as shown in Figure 1. Saline-injected mice served as controls and had no detectable antibody response. The IgG response in the mice treated with J5dLPS had a mean of 6395 +/- 2286 (SE) ODUs/ mL; whereas, the saline-treated mice had a mean IgG response of 0 +/- 0 ODUs/mL; the p-value between the two groups was p< 0.0062.