Xingdong Yang

High dose and low dose Lactobacillus acidophilus exerted differential immune modulating effects on T cell immune responses induced by an oral human rotavirus vaccine in gnotobiotic pigs

BACKGROUND Strain-specific effects of probiotics in pro- or anti-inflammatory immune responses have been well recognized. Several proinflammatory Lactobacillus strains have been shown to act as adjuvants to enhance the immunogenicity of vaccines. However, dose effects of probiotics in modulating immune responses are not clearly understood. This study examined the dose effects of Lactobacillus acidophilus (LA) NCFM strain on T cell immune responses to rotavirus vaccination in a gnotobiotic (Gn) pig model. METHODS Frequencies of IFN-γ producing CD4+ and CD8+ T cell and IL-10 and TGF-β producing CD4+CD25+ and CD4+CD25- regulatory T (Treg) cell responses were determined in the intestinal and systemic lymphoid tissues of Gn pigs vaccinated with an oral human rotavirus vaccine in conjunction with low dose (5 feedings; up to 10(6) colony forming units [CFU]/dose) or high dose (14 feedings; up to 10(9)CFU/dose) or without LA feeding. RESULTS Low dose LA significantly promoted IFN-γ producing T cell responses and down-regulated Treg cell responses and their TGF-β and IL-10 productions in all the tissues compared to the high dose LA and control groups. To the contrary, high dose LA increased the frequencies of Treg cells in most of the tissues compared to the control groups. The dose effects of LA on IFN-γ producing T cell and CD4+CD25- Treg cell immune responses were similar in the intestinal and systemic lymphoid tissues and were independent from the vaccination. CONCLUSION Thus the same probiotic strain in different doses can either promote or suppress IFN-γ producing T cell or Treg cell immune responses. These findings have significant implications in the use of probiotic lactobacilli as immunostimulatory versus immunoregulatory agents. Probiotics can be ineffective or even detrimental if not used at the optimal dosage for the appropriate purposes.

Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 109 colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 106 CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.

Lactobacillus rhamnosus GG on Rotavirus-Induced Injury of Ileal Epithelium in Gnotobiotic Pigs

Objective: The aim of this study was to study the effect of continued Lactobacillus rhamnosus GG strain (LGG) feeding on rotavirus gastroenteritis in the gnotobiotic (Gn) pig model of virulent human rotavirus (HRV) infection. Methods: Gn pigs were assigned to treatment groups: mock control, LGG only, HRV only, or LGG plus HRV. Nine days before HRV inoculation (3 days of age), pigs were fed LGG with a daily dose increase of 10-fold from 103 to 1012 colony-forming units (CFU). The 1012 CFU/dose of LGG feeding continued until post-HRV inoculation day (PID) 6. Clinical sign (diarrhea), rotavirus fecal shedding, histopathology of the ileum, adherent junction and tight junction protein expression in the ileal epithelial cells, mucin production in the large and small intestinal contents, and serum cytokine responses from PID 2 to 6 were examined and compared among the treatment groups. Results: Clinically, the percentage of pigs developing diarrhea, the mean duration of diarrhea, and the mean cumulative fecal scores were lower in the LGG fed pigs compared to the nonfed pigs after HRV inoculation. LGG partially protected ileal epithelium against HRV-induced compensatory increases of the adherent junction protein &agr;-catenin and &bgr;-catenin, tight junction protein occludin, claudin-3 and claudin-4, and leak protein claudin-2. LGG promoted mucin production because the mucin levels in the large intestinal contents of the LGG+HRV pigs were significantly higher than the HRV-only pigs on PID 2. Additionally, LGG maintained the anti-inflammatory cytokine transforming growth factor-&bgr; level in serum after HRV infection. Conclusions: LGG is moderately effective for ameliorating rotavirus diarrhea by partially preventing injuries to the epithelium.

Median infectious dose of human norovirus GII.4 in gnotobiotic pigs is decreased by simvastatin treatment and increased by age

Human noroviruses (NoVs), a major cause of viral gastroenteritis, are difficult to study due to the lack of a cell-culture and a small-animal model. Pigs share with humans the types A and H histo-blood group antigens on the intestinal epithelium and have been suggested as a potential model for studies of NoV pathogenesis, immunity and vaccines. In this study, the effects of age and a cholesterol-lowering drug, simvastatin, on the susceptibility of pigs to NoV infection were evaluated. The median infectious dose (ID50) of a genogroup II, genotype 4 (GII.4) 2006b variant was determined. The ID50 in neonatal (4-5 days of age) pigs was ≤2.74×10(3) viral RNA copies. In older pigs (33-34 days of age), the ID50 was 6.43×10(4) but decreased to <2.74×10(3) in simvastatin-fed older pigs. Evidence of NoV infection was obtained by increased virus load in the intestinal contents, cytopathological changes in the small intestine, including irregular microvilli, necrosis and apoptosis, and detection of viral antigen in the tip of villi in duodenum. This GII.4 variant was isolated in 2008 from a patient from whom a large volume of stool was collected. GII.4 NoVs are continuously subjected to selective pressure by human immunity, and antigenically different GII.4 NoV variants emerge every 1-2 years. The determination of the ID50 of this challenge virus is valuable for evaluation of protection against different GII.4 variants conferred by NoV vaccines in concurrence with other GII.4 variants in the gnotobiotic pig model.

Probiotics and virulent human rotavirus modulate the transplanted human gut microbiota in gnotobiotic pigs

We generated a neonatal pig model with human infant gut microbiota (HGM) to study the effect of a probiotic on the composition of the transplanted microbiota following rotavirus vaccination and challenge. All the HGM-transplanted pigs received two doses of an oral attenuated rotavirus vaccine. The gut microbiota of vaccinated pigs were investigated for effects of Lactobacillus rhamnosus GG (LGG) supplement and homotypic virulent human rotavirus (HRV) challenge. High-throughput sequencing of V4 region of 16S rRNA genes demonstrated that HGM-transplanted pigs carried microbiota similar to that of the C-section delivered baby. Firmicutes and Proteobacteria represented over 98% of total bacteria in the human donor and the recipient pigs. HRV challenge caused a phylum-level shift from Firmicutes to Proteobacteria. LGG supplement prevented the changes in microbial communities caused by HRV challenge. In particular, members of Enterococcus in LGG-supplemented pigs were kept at the baseline level, while they were enriched in HRV challenged pigs. Taken together, our results suggested that HGM pigs are valuable for testing the microbiota’s response to probiotic interventions for treating infantile HRV infection.

Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs

ABSTRACT Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and virus-like particles (VLPs) against NoV infection and disease and the T cell responses to these treatments. Pigs either were vaccinated intranasally with GII.4/1997 NoV (VA387)-derived P particles or VLPs or were inoculated orally with a GII.4/2006b NoV variant. At postinoculation day (PID) 28, pigs either were euthanized or were challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% homologous protection against virus shedding, while the P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in the duodenum and are positively correlated with T cell expansion in the ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher numbers of activated CD4+ T cells in all tissues, gamma interferon-producing (IFN-γ+) CD8+ T cells in the duodenum, regulatory T cells (Tregs) in the blood, and transforming growth factor β (TGF-β)-producing CD4+ CD25− FoxP3+ Tregs in the spleen postchallenge, indicating that P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development. IMPORTANCE The norovirus (NoV) P particle is a vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. P particles can be easily produced in Escherichia coli at high yields and thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated, for the first time, the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses by using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the VLP vaccine in pigs (60%) and to the homologous protective efficacy of the VLP vaccine in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine holds promise for reducing the disease burden and mortality.

Dual functions of Lactobacillus acidophilus NCFM as protection against rotavirus diarrhea.

Objective: The aim of the study was to examine the dose effects of Lactobacillus acidophilus (LA) NCFM strain on rotavirus-specific antibody and B-cell responses in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV). Methods: Pigs were inoculated with AttHRV vaccine in conjunction with high-dose LA (14 doses, total 2.2 × 109 colony-forming units [CFU]), intermediate-dose LA (MidLA) (9 doses, total 3.2 × 106 CFU), low-dose LA (LoLA) (5 doses, total 2.1 × 106 CFU), or without LA feeding. Protection against rotavirus shedding and diarrhea was assessed upon challenge with a virulent HRV. Rotavirus-specific immunoglobulin A (IgA) and IgG antibodies in serum and rotavirus-specific IgA and IgG antibody-secreting cells (ASCs) and memory B cells in ileum, spleen, and blood of the pigs were measured and compared among treatment groups. Results: The MidLA, but not high-dose LA or LoLA, significantly reduced rotavirus diarrhea (MidLA-only group) and significantly improved the protection conferred by AttHRV vaccine (MidLA + AttHRV group). Associated with the increased protection, MidLA significantly enhanced rotavirus-specific antibody, ASCs, and memory B-cell responses to AttHRV vaccine. High-dose LA or LoLA did not enhance virus-specific antibody and ASC responses, and hence did not improve the vaccine efficacy. Conclusions: These findings highlight the importance of dose selection and indicate that certain specific lactobacilli strains at the appropriate dose have the dual function of reducing rotavirus diarrhea and enhancing the immunogenicity and protective efficacy of rotavirus vaccines.

High protective efficacy of rice bran against human rotavirus diarrhea via enhancing probiotic growth, gut barrier function, and innate immunity.

Previously, we showed that rice bran (RB) was able to reduce human rotavirus (HRV) diarrhea in gnotobiotic pigs. Here, we investigated its effect on the growth of diarrhea-reducing probiotic Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle (EcN), and the resulting effects on HRV diarrhea, gut epithelial health, permeability and innate immune responses during virulent HRV challenge. On 3, 5, and 7 days of age pigs were inoculated with 2 × 104 colony-forming-units LGG+EcN to initiate colonization. Daily RB supplementation (replacing 10% calorie intake) was started at 5 days of age and continued until euthanasia. A subset of pigs in each group was challenged orally with 105 focus-forming-units of virulent HRV at 33 days of age. RB completely prevented HRV diarrhea in LGG+EcN colonized pigs. RB significantly promoted the growth of both probiotic strains in the gut (~5 logs) and increased the body-weight-gain at 4–5 weeks of age compared to non-RB group. After HRV challenge, RB-fed pigs had significantly lower ileal mitotic index and villus width, and significantly increased intestinal IFN-γ and total IgA levels compared to non-RB group. Therefore, RB plus LGG+EcN colonization may represent a highly effective therapeutic approach against HRV and potentially a variety of other diarrhea-inducing enteric pathogens.

Probiotic Lactobacillus rhamnosus GG mono-association suppresses human rotavirus-induced autophagy in the gnotobiotic piglet intestine

BackgroundHuman rotavirus (HRV) is the most important cause of severe diarrhea in infants and young children. Probiotic Lactobacillus rhamnosus GG (LGG) reduces rotavirus infection and diarrhea. However, the molecular mechanisms of LGG-mediated protection from rotavirus infection are poorly understood. Autophagy plays an essential role in responses to microbial pathogens. However, the role of autophagy in HRV infection and LGG treatment is unknown. We hypothesize that rotavirus gastroenteritis activates autophagy and that LGG suppresses virus-induced autophagy and prevents intestinal damage in infected piglets.MethodsWe used LGG feeding to combat viral gastroenteritis in the gnotobiotic pig model of virulent HRV infection.ResultsWe found that LGG feeding did not increase autophagy, whereas virus infection induced autophagy in the piglet intestine. Virus infection increased the protein levels of the autophagy markers ATG16L1 and Beclin-1 and the autophagy regulator mTOR. LGG treatment during viral gastroenteritis reduced autophagy marker expression to normal levels, induced apoptosis and partially prevented virus-induced tissue damage.ConclusionOur study provides new insights into virus-induced autophagy and LGG suppression of uncontrolled autophagy and intestinal injury. A better understanding of the antiviral activity of LGG will lead to novel therapeutic strategies for infant infectious diseases.

CD4+ CD25− FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

The distribution and dynamic changes of CD4+ CD25+ FoxP3+ and CD4+ CD25− FoxP3+ regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post‐inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue‐residing Treg cells, and decreased the frequencies of interleukin‐10 (IL‐10) and transforming growth factor‐β (TGF‐β) producing CD4+ CD25− Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL‐10 and TGF‐β producing CD4+ CD25− Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV‐caused diarrhoea (r = −1, P < 0·0001). Hence, higher frequencies of functional CD4+ CD25− Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4+ CD25− Treg cells over CD4+ CD25+ Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4+ CD25− Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF‐β producing Treg cells post‐challenge. The reduced suppressive effect of Treg cells in AttHRV‐vaccinated pigs would unleash effector/memory T‐cell activation upon challenge. Preserving TGF‐β producing CD4+ CD25− Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T‐cell responses after primary rotavirus infection/vaccination and challenge.