Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model.
ABSTRACT: Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFN?) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFN? (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.
Project description:Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-? and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-?, splenic and circulatory IFN-?-producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV.
Project description:The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.
Project description:B cells play a key role in generation of protective immunity against rotavirus infection, a major cause of gastroenteritis in children. Current RV vaccines are less effective in developing countries compared to developed countries. Commensals/probiotics influence mucosal immunity, but the role of early gut colonizing bacteria in modulating intestinal B cell responses to RV vaccines is largely unknown. We co-colonized neonatal gnotobiotic pigs, the only animal model susceptible to HRV diarrhea, with 2 dominant bacterial species present in the gut of breastfed infants, Lactobacillus rhamnosus strain GG and Bifidobacterium animalis lactis Bb12 to evaluate their impact on B cell responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine. Following HRV challenge, probiotic-colonized, AttHRV vaccinated piglets had significantly lower fecal scores and reduced HRV shedding titers compared to uncolonized, AttHRV vaccinated pigs. The reduction in HRV diarrhea was significantly correlated with higher intestinal IgA HRV antibody titers and intestinal HRV-specific IgA antibody secreting cell (ASC) numbers in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs. The significantly higher small intestinal HRV IgA antibody responses coincided with higher IL-6, IL-10 and APRIL responses of ileal mononuclear cells (MNCs) and the immunomodulatory effects of probiotics genomic DNA on TGF-β and IL-10 responses. However, serum RV IgG antibody titers and total IgG titers were significantly lower in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs, both pre- and post-challenge. In summary, LGG and Bb12 beneficially modulated intestinal B cell responses to HRV vaccine.
Project description:Genetically modified pigs have become available recently. In this study, we established the gnotobiotic pig model of human rotavirus (HRV) infection using cloned pigs with homozygous disruption in the gene encoding immunoglobulin heavy chain (HCKO), which totally impairs B-cell development. To clarify importance of B cells and cytotoxic T cells in rotavirus immunity, CD8 cells in a subset of the pigs were depleted by injecting antipig CD8 antibodies and the immune phenotypes of all pigs were examined. HCKO pigs, CD8 cell-depleted HCKO pigs, and wild-type (WT) pigs were vaccinated with an attenuated HRV vaccine and challenged with virulent HRV. Protection against HRV infection and diarrhea was assessed postchallenge and detailed T-cell subset responses were determined pre- and postchallenge. Significantly longer duration of virus shedding was seen in vaccinated HCKO pigs than in WT pigs, indicating the importance of B cells in vaccine-induced protective immunity. Vaccinated HCKO/CD8(-) pigs shed significantly higher number of infectious virus than WT pigs and non-CD8-depleted HCKO pigs, indicating the importance of CD8 T cells in controlling virus replication. Therefore, both B cells and CD8 T cells play an important role in the protection against rotavirus infection. HCKO and HCKO/CD8(-) pigs did not differ significantly in diarrhea and virus shedding postchallenge; increased CD4 and CD8(-) ?? T-cell responses probably compensated partially for the lack of CD8 T cells. This study demonstrated that HCKO pigs can serve as a valuable model for dissection of protective immune responses against viral infections and diseases.
Project description:Breast milk (colostrum [col]/milk) components and gut commensals play important roles in neonatal immune maturation, establishment of gut homeostasis and immune responses to enteric pathogens and oral vaccines. We investigated the impact of colonization by probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) with/without col/milk (mimicking breast/formula fed infants) on B lymphocyte responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine in a neonatal gnotobiotic pig model. Col/milk did not affect probiotic colonization in AttHRV vaccinated pigs. However, unvaccinated pigs fed col/milk shed higher numbers of probiotic bacteria in feces than non-col/milk fed colonized controls. In AttHRV vaccinated pigs, col/milk feeding with probiotic treatment resulted in higher mean serum IgA HRV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to col/milk fed, non-colonized vaccinated pigs. In vaccinated pigs without col/milk, probiotic colonization did not affect IgA HRV antibody titers, but serum IgG HRV antibody titers and gut IgG ASC numbers were lower, suggesting that certain probiotics differentially impact HRV vaccine responses. Our findings suggest that col/milk components (soluble mediators) affect initial probiotic colonization, and together, they modulate neonatal antibody responses to oral AttHRV vaccine in complex ways.
Project description: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.
Project description:Vitamin A deficiencies and insufficiencies are widespread in developing countries, and may be gaining prevalence in industrialized nations. To combat vitamin A deficiency (VAD), the World Health Organization (WHO) recommends high-dose vitamin A supplementation (VAS) in children 6-59 months of age in locations where VAD is endemic. This practice has significantly reduced all-cause death and diarrhea-related mortalities in children, and may have in some cases improved immune responses toward pediatric vaccines. However, VAS studies have yielded conflicting results, perhaps due to influences of baseline vitamin A levels on VAS efficacy, and due to cross-regulation between vitamin A and related nuclear hormones. Here we provide a brief review of previous pre-clinical and clinical data, showing how VAD and VAS affect immune responses, vaccines, and infectious diseases. We additionally present new results from a VAD mouse model. We found that when VAS was administered to VAD mice at the time of vaccination with a pneumococcal vaccine (Prevnar-13), pneumococcus (T4)-specific antibodies were significantly improved. Preliminary data further showed that after challenge with Streptococcus pneumoniae, all mice that had received VAS at the time of vaccination survived. This was a significant improvement compared to vaccination without VAS. Data encourage renewed attention to vitamin A levels, both in developed and developing countries, to assist interpretation of data from vaccine research and to improve the success of vaccine programs.
Project description:We have shown previously that vitamin A deficiency (VAD) leads to the decreased expression of gene products that are specifically synthesized by mature neurons in the olfactory epithelium (OE) of adult rats. These results support the hypothesis that retinoic acid, a derivative of vitamin A, is required for neurogenesis and neuron replacement in vivo. VAD does not cause gross degeneration of the OE, raising the question: what types of cells continue to populate VAD OE? In this study, we compared the cell densities of VAD and VA-sufficient (VAS) OE and investigated whether cell proliferation is upregulated in VAD OE. The results show that (1) total cell number in VAD and VAS OE are comparable; (2) localized areas of hyperplasia are present in the basal regions of VAD, but not VAS, OE; (3) there is a substantial increase in the number of PCNA (proliferating cell nuclear antigen) positive cells in the basal region of VAD OE relative to VAS OE; and (4) there is a relative increase in the levels of mRNA encoding the transcription factor, MASH I, in VAD OE. We conclude that reduced availability of vitamin A derivatives, such as retinoic acid, leads to a loss of control over proliferation, hyperplasia, and increased numbers of pro-neural cells in vivo.
Project description:Observational studies in human suggest involvement of vitamin A/retinoic acid (RA) signaling in the regulation of airway smooth muscle (ASM) function, but the precise mechanisms by which RA impacts ASM phenotype is not clear. Here, we generated trascriptional profiles from two different models of RA-sufficient and RA-deficient mouse ASM in order to determine the molecular targets of RA in ASM (VAS/VAD, CTR/BMS) Mouse ASMs were isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. VAS mice were given normal, vitamin A-sufficient diet (15 IU vitamin/g) while the VAD mice were given 3 days of vitamin A-deficient diet (< 0.1 IU vitamin/g). CTR/BMS mouse ASMs were also isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. CTR mice were given 3 days of powdered, vitamin A sufficient diet mixed with vehicles (corn oil) while the BMS diet were given 3 days of powdered, vitamin A sufficient diet mixed with a potent pan-RA-receptor antagonist (BMS, 5 mcg/g). Overall design: Mouse ASMs from 3 mice in each experimental condition were collected and pooled for microarray analysis.
Project description:BACKGROUND:Low efficacy of rotavirus (RV) vaccines in developing African and Asian countries, where malnutrition is prevalent, remains a major concern and a challenge for global health. METHODS:To understand the effects of protein malnutrition on RV vaccine efficacy, we elucidated the innate, T cell and cytokine immune responses to attenuated human RV (AttHRV) vaccine and virulent human RV (VirHRV) challenge in germ-free (GF) pigs or human infant fecal microbiota (HIFM) transplanted gnotobiotic (Gn) pigs fed protein-deficient or -sufficient bovine milk diets. We also analyzed serum levels of tryptophan (TRP), a predictor of malnutrition, and kynurenine (KYN). RESULTS:Protein-deficient pigs vaccinated with oral AttHRV vaccine had lower protection rates against diarrhea post-VirHRV challenge and significantly increased fecal virus shedding titers (HIFM transplanted but not GF pigs) compared with their protein-sufficient counterparts. Reduced vaccine efficacy in protein-deficient pigs coincided with altered serum IFN-?, TNF-?, IL-12 and IFN-? responses to oral AttHRV vaccine and the suppression of multiple innate immune parameters and HRV-specific IFN-? producing T cells post-challenge. In protein-deficient HIFM transplanted pigs, decreased serum KYN, but not TRP levels were observed throughout the experiment, suggesting an association between the altered TRP metabolism and immune responses. CONCLUSION:Collectively, our findings confirm the negative effects of protein deficiency, which were exacerbated in the HIFM transplanted pigs, on innate, T cell and cytokine immune responses to HRV and on vaccine efficacy, as well as on TRP-KYN metabolism.