4. Gut flora & adaptive immunity
CD4+ T cell subset differentiation (Th1, Th2, Th17,
Treg): abnormal in germ free mice and skewed by
specific commensal bacteria (e.g. SFB, Bifidobacteria)
or their products (polysaccharide A, ATP, CpG)
Expansion and activation of cytolytic CD8+ T cells may
be affected by composition of enteric microbiota
5. Gut flora alterations
Changes in diet (e.g. weaning)
Immune responses to enteric pathogens
Antibiotics
Number of antimicrobial drugs dispensed per year
Finkelstein JA et al (2000) Arch Pediatr Adolesc Med 154: 395-400
6. Hypothesis: Altering gut flora
will impact vaccine efficacy
Reproducible alteration of flora
Clinically significant disease
Relevant murine vaccination/disease model
7. West Nile virus (WNV)
Vaccine antigen: E protein plus alum adjuvant
Systemic (ip) administration
Murine disease challenge model
15. Antibiotic protcol
3 week old female mice (C57Bl/6) to SPF housing
Duplicate cages randomized to treatment x 1 wk
Control (autoclaved chow/water)
Vancomycin/Norfloxacin/Metronidazole
Neomycin/Clindamycin
16. Antibiotic protcol
3 week old female mice (C57Bl/6) to SPF housing
Duplicate cages randomized to treatment x 1 wk
Control (autoclaved chow/water)
Vancomycin/Norfloxacin/Metronidazole
Neomycin/Clindamycin
Mice off antibiotics x 1 week
Conventionalized (complete fecal flora)
Not conventionalized
33. Summary
Short term antibiotic exposure is associated with an
attenuated response to systemic vaccination
Mechanism: defects in antigen presentation?
Mechanism: missing OTU/functionality v. taxonomic imbalance?
Vaccine response can be normalized by returning gut
flora to a phylogenetic distribution characteristic of
control mice
Can a defined probiotic normalize vaccine response?
Can specific adjuvants provide this functionality?
Vaccines are a cornerstone of public health policy. This table clearly shows how the adoption of childhood vaccination recommendations has reduced morbidity for a number of vaccine preventable diseases between the beginning and end of the last century. Nonetheless, we know that vaccines are not uniformly effective in all hosts.\n
Some of this host variability is likely due to factors such as age, nutritional status and even genetic variation. As this meeting has clearly illustrated, however, we also need to consider whether variation in a host’s microbiome might influence their response to vaccination.\n
Work from many investigators has now clearly established links between the gut microbiome and the development of adaptive immunity. Differentiation of CD4 T cell subsets does not occur normally in germ free mice, and can be skewed....\nAlthough CD8 T cells differentiate in the absence of the gut microflora, their expansion &...\n
Although humans are obviously never germ free, we clearly experience events that dramatically shift the composition of our microbiome. These include dietary changes, particularly those that accompany weaning, innate immune responses that we mount to infections, and of course antibiotic use. This graph shows how frequently children under the age of three are dispensed antibiotics in the United States--and we can see that some 75% received one or more courses of antimicrobials in the year examined in this study\n
Given this background, we wanted to test whether such alterations in gut flora might alter the response to vaccination. We carried out our experiments in mice, first defining a strategy that would reproducibly alter their gut flora and then testing how such alterations affected their response to vaccine and subsequent pathogen challenge. We were particularly interested in murine models for clinically significant human diseases and therefore initially focused these studies on the flavivirus West Nile\n
West Nile Virus has caused substantial human morbidity and mortality in the US during the past decade. Although there is no human vaccine approved for WN, there is a vaccine that has been used successfully in horses in the US. The vaccine is the purified envelope protein, which we administered systemically with alum adjuvant at a dose that protects mice against challenge with live virus.\n
So the general strategy of our experiments was to take mice, expose them to antibiotics for a week, then assess...\n
So the general strategy of our experiments was to take mice, expose them to antibiotics for a week, then assess...\n
So the general strategy of our experiments was to take mice, expose them to antibiotics for a week, then assess...\n
So the general strategy of our experiments was to take mice, expose them to antibiotics for a week, then assess...\n
We purchased and housed control mice, as well as those randomized to receive either vancomycin , norfloxacin and metronidazole OR neomycin and clindamycin in specific pathogen free housing, where they were fed autoclaved water and chow. The mice received antibiotics orally for one week, then were allowed to recover for an additional week. We collected stool at the end of week 1 and week 2 to analyze fecal bacterial counts, and analyzed OTU composition by pyrosequencing of the V3 region of 16s rRNA. In some experiments we attempted to alter the gut microflora by exposing mice to complete fecal flora from control animals during the recovery week, and I will call these animals “conventionalized” during the remainder of the talk. \n
We purchased and housed control mice, as well as those randomized to receive either vancomycin , norfloxacin and metronidazole OR neomycin and clindamycin in specific pathogen free housing, where they were fed autoclaved water and chow. The mice received antibiotics orally for one week, then were allowed to recover for an additional week. We collected stool at the end of week 1 and week 2 to analyze fecal bacterial counts, and analyzed OTU composition by pyrosequencing of the V3 region of 16s rRNA. In some experiments we attempted to alter the gut microflora by exposing mice to complete fecal flora from control animals during the recovery week, and I will call these animals “conventionalized” during the remainder of the talk. \n
We purchased and housed control mice, as well as those randomized to receive either vancomycin , norfloxacin and metronidazole OR neomycin and clindamycin in specific pathogen free housing, where they were fed autoclaved water and chow. The mice received antibiotics orally for one week, then were allowed to recover for an additional week. We collected stool at the end of week 1 and week 2 to analyze fecal bacterial counts, and analyzed OTU composition by pyrosequencing of the V3 region of 16s rRNA. In some experiments we attempted to alter the gut microflora by exposing mice to complete fecal flora from control animals during the recovery week, and I will call these animals “conventionalized” during the remainder of the talk. \n
We enumerated bacteria in stool samples by staining with Sybr Green and then counting the organisms microscopically, since we had difficulty quantitatively recovering strict anaerobes by culture methods. As you can see, each antibiotic regimen leads to a several-fold decrease in the number of stool bacteria by the end of week 1, which remains stable over the recovery week. If we conventionalize mice after either treatment regimen, we increase stool counts back to control levels. So the changes in bacterial number are similar between these two antibiotic regimens, but the shifts in microbiome composition are not, as we can see on the next slide.\n
Weaned mice, like humans, have a gut microflora in which bacteroidetes predominate. Administration of either antibiotic regimen dramatically changed this picture but in completely distinct ways. So as you can see, proteobacteria become dominant in neomycin/clinda treated mice, while Vanco/norflox/metronidazole, or VNM mice shift to a microbiome dominated by firmicutes, specifically Lactobacillus species. These shifts are stable over the next week in the absence of conventionalization. If we expose Neo/Clinda mice to normal fecal flora, we slightly shift the balance between proteobacteria and firmicutes, but can’t restore Bacteroidetes. On the other hand, VNM mice can be shifted back to a microbiome composition in which Bacteroidetes predominates.\n
Weaned mice, like humans, have a gut microflora in which bacteroidetes predominate. Administration of either antibiotic regimen dramatically changed this picture but in completely distinct ways. So as you can see, proteobacteria become dominant in neomycin/clinda treated mice, while Vanco/norflox/metronidazole, or VNM mice shift to a microbiome dominated by firmicutes, specifically Lactobacillus species. These shifts are stable over the next week in the absence of conventionalization. If we expose Neo/Clinda mice to normal fecal flora, we slightly shift the balance between proteobacteria and firmicutes, but can’t restore Bacteroidetes. On the other hand, VNM mice can be shifted back to a microbiome composition in which Bacteroidetes predominates.\n
Weaned mice, like humans, have a gut microflora in which bacteroidetes predominate. Administration of either antibiotic regimen dramatically changed this picture but in completely distinct ways. So as you can see, proteobacteria become dominant in neomycin/clinda treated mice, while Vanco/norflox/metronidazole, or VNM mice shift to a microbiome dominated by firmicutes, specifically Lactobacillus species. These shifts are stable over the next week in the absence of conventionalization. If we expose Neo/Clinda mice to normal fecal flora, we slightly shift the balance between proteobacteria and firmicutes, but can’t restore Bacteroidetes. On the other hand, VNM mice can be shifted back to a microbiome composition in which Bacteroidetes predominates.\n
Weaned mice, like humans, have a gut microflora in which bacteroidetes predominate. Administration of either antibiotic regimen dramatically changed this picture but in completely distinct ways. So as you can see, proteobacteria become dominant in neomycin/clinda treated mice, while Vanco/norflox/metronidazole, or VNM mice shift to a microbiome dominated by firmicutes, specifically Lactobacillus species. These shifts are stable over the next week in the absence of conventionalization. If we expose Neo/Clinda mice to normal fecal flora, we slightly shift the balance between proteobacteria and firmicutes, but can’t restore Bacteroidetes. On the other hand, VNM mice can be shifted back to a microbiome composition in which Bacteroidetes predominates.\n
Weaned mice, like humans, have a gut microflora in which bacteroidetes predominate. Administration of either antibiotic regimen dramatically changed this picture but in completely distinct ways. So as you can see, proteobacteria become dominant in neomycin/clinda treated mice, while Vanco/norflox/metronidazole, or VNM mice shift to a microbiome dominated by firmicutes, specifically Lactobacillus species. These shifts are stable over the next week in the absence of conventionalization. If we expose Neo/Clinda mice to normal fecal flora, we slightly shift the balance between proteobacteria and firmicutes, but can’t restore Bacteroidetes. On the other hand, VNM mice can be shifted back to a microbiome composition in which Bacteroidetes predominates.\n
We next went on to measure response to vaccination in our control and antibiotic exposed animals. \n
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Weight loss correlates well with viremia.\n
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LEfSe analysis with p=0.01 carried out using John Bruce’s data sets (min length 100), with NC included in the non-responders. (39: LDA Effect size on data 38)\n
LEfSe analysis with p=0.01 carried out using John Bruce’s data sets (min length 100), with NC included in the non-responders. (39: LDA Effect size on data 38)\n
LEfSe analysis carried out on min180 RDP data set (processed by myself), using p=0.01.\n
LEfSe analysis carried out on min180 RDP data set (processed by myself), using p=0.01.\n
LEfSe analysis carried out on min180 RDP data set (processed by myself), using p=0.05.\n
LEfSe analysis carried out on min180 RDP data set (processed by myself), using p=0.05.\n
