Human Rotavirus Diarrhea Research Articles

MRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea.

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

Rice Bran and Probiotics Alter the Porcine Large Intestine and Serum Metabolomes for Protection against Human Rotavirus Diarrhea.

Human rotavirus (HRV) is a leading cause of severe childhood diarrhea, and there is limited vaccine efficacy in the developing world. Neonatal gnotobiotic pigs consuming a prophylactic synbiotic combination of probiotics and rice bran (Pro+RB) did not exhibit HRV diarrhea after challenge. Multiple immune, gut barrier protective, and anti-diarrheal mechanisms contributed to the prophylactic efficacy of Pro+RB when compared to probiotics (Pro) alone. In order to understand the molecular signature associated with diarrheal protection by Pro+RB, a global non-targeted metabolomics approach was applied to investigate the large intestinal contents and serum of neonatal gnotobiotic pigs. The ultra-high performance liquid chromatography-tandem mass spectrometry platform revealed significantly different metabolites (293 in LIC and 84 in serum) in the pigs fed Pro+RB compared to Pro, and many of these metabolites were lipids and amino acid/peptides. Lipid metabolites included 2-oleoylglycerol (increased 293.40-fold in LIC of Pro+RB, p = 3.04E-10), which can modulate gastric emptying, andhyodeoxycholate (decreased 0.054-fold in the LIC of Pro+RB, p = 0.0040) that can increase colonic mucus production to improve intestinal barrier function. Amino acid metabolites included cysteine (decreased 0.40-fold in LIC, p = 0.033, and 0.62-fold in serum, p = 0.014 of Pro+RB), which has been found to reduce inflammation, lower oxidative stress and modulate mucosal immunity, and histamine (decreased 0.18-fold in LIC, p = 0.00030, of Pro+RB and 1.57-fold in serum, p = 0.043), which modulates local and systemic inflammatory responses as well as influences the enteric nervous system. Alterations to entire LIC and serum metabolic pathways further contributed to the anti-diarrheal and anti-viral activities of Pro+RB such as sphingolipid, mono/diacylglycerol, fatty acid, secondary bile acid, and polyamine metabolism. Sphingolipid and long chain fatty acid profiles influenced the ability of HRV to both infect and replicate within cells, suggesting that Pro+RB created a protective lipid profile that interferes with HRV activity. Polyamines act on enterocyte calcium-sensing receptors to modulate intracellular calcium levels, and may directly interfere with rotavirus replication. These results support that multiple host and probiotic metabolic networks, notably those involving lipid and amino acid/peptide metabolism, are important mechanisms through which Pro+RB protected against HRV diarrhea in neonatal gnotobiotic pigs.

Unraveling the Differences between Gram-Positive and Gram-Negative Probiotics in Modulating Protective Immunity to Enteric Infections.

The role of intestinal microbiota and probiotics in prevention and treatment of infectious diseases, including diarrheal diseases in children and animal models, is increasingly recognized. Intestinal commensals play a major role in development of the immune system in neonates and in shaping host immune responses to pathogens. Lactobacilli spp. and Escherichia coli Nissle 1917 are two probiotics that are commonly used in children to treat various medical conditions including human rotavirus diarrhea and inflammatory bowel disease. Although the health benefits of probiotics have been confirmed, the specific effects of these established Gram-positive (G+) and Gram-negative (G−) probiotics in modulating immunity against pathogens and disease are largely undefined. In this review, we discuss the differences between G+ and G− probiotics/commensals in modulating the dynamics of selected infectious diseases and host immunity. These probiotics modulate the pathogenesis of infectious diseases and protective immunity against pathogens in a species- and strain-specific manner. Collectively, it appears that the selected G− probiotic is more effective than the various tested G+ probiotics in enhancing protective immunity against rotavirus in the gnotobiotic piglet model.

High Protective Efficacy of Probiotics and Rice Bran against Human Norovirus Infection and Diarrhea in Gnotobiotic Pigs.

Probiotics have been recognized as vaccine adjuvants and therapeutic agents to treat acute gastroenteritis in children. We previously showed that rice bran (RB) reduced human rotavirus diarrhea in gnotobiotic pigs. Human noroviruses (HuNoVs) are the major pathogens causing non-bacterial acute gastroenteritis worldwide. In this study, Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) were first screened for their ability to bind HuNoV P particles and virions derived from clinical samples containing HuNoV genotype GII.3 and GII.4, then the effects of LGG+EcN and RB on HuNoV infection and diarrhea were investigated using the gnotobiotic pig model. While LGG+EcN colonization inhibited HuNoV shedding, probiotic cocktail regimens in which RB feeding started 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized gnotobiotic pigs exhibited high protection against HuNoV diarrhea and shedding, characterized by significantly reduced incidence (89 versus 20%) and shorter mean duration of diarrhea (2.2 versus 0.2 days), as well as shorter mean duration of virus shedding (3.2 versus 1.0 days). In both probiotic cocktail groups, the diarrhea reduction rates were 78% compared with the control group, and diarrhea severity was reduced as demonstrated by the significantly lower cumulative fecal scores. The high protective efficacy of the probiotic cocktail regimens was attributed to stimulation of IFN-γ+ T cell responses, increased production of intestinal IgA and IgG, and maintenance of healthy intestinal morphology (manifested as longer villi compared with the control group). Therefore, probiotic cocktail regimens containing LGG+EcN and RB may represent highly efficacious strategies to prevent and treat HuNoV gastroenteritis, and potentially other human enteric pathogens.

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.

Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model

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.

Escherichia coli Nissle 1917 colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model (VIR7P.1064)

Abstract Microbiota regulate gut immune homeostasis, but the role of gram positive (G+)/negative (G-) commensals/probiotics in modulating human rotavirus (HRV) diarrhea and immunity is largely undefined. Gnotobiotic (Gn) piglets, the only animal model susceptible to HRV diarrhea, were colonized with G- Escherichia coli Nissle 1917 (EcN), G+ Lactobacillus rhamnosus strain GG (LGG) or LGG+EcN and challenged with virulent HRV (VirHRV) to assess probiotic effects on HRV pathogenesis and immunity. EcN+VirHRV piglets had significantly lower mean peak virus shedding titers and significantly reduced mean cumulative fecal scores and duration of diarrhea compared to LGG+VirHRV or control (uncolonized)+VirHRV piglets. LGG+VirHRV pigs had lower, but statistically similar fecal scores compared to control+VirHRV pigs. Combining LGG+EcN did not moderate VirHRV shedding or diarrhea more than EcN alone. Coinciding with reduced HRV infection, EcN+VirHRV piglets had significantly lower small intestinal (SI) HRV IgA antibody titers and HRV IgA and IgG antibody secreting cells (SC) compared to control+VirHRV piglets. Though not significantly different, SI HRV IgA antibody titers were 2-4-fold lower in the LGG+EcN+VirHRV and LGG+VirHRV piglets. Notably EcN+VirHRV piglets had higher SI total IgA levels and IgA SC compared to LGG+VirHRV piglets. Our results indicate that EcN alone was more effective than LGG or LGG+EcN in ameliorating HRV diarrhea and modulating B cell responses in a Gn piglet disease model.

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

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 (IFNα) 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 IFNγ (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.

Prenatally Acquired Vitamin A Deficiency Alters Innate Immune Responses to Human Rotavirus in a Gnotobiotic Pig Model

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A-sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000 IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEβ7) expressing DCs in VAS versus VAD piglets postchallenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post-VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α levels in VAD pigs that coincided with decreased TLR3(+) MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.

Prospective study of community-acquired rotavirus infection

We determined titers of group A rotavirus common antibodies and neutralizing antibodies against serotypes 1 to 4 of prototype human rotavirus (HRV) in cord blood and serum specimens obtained from 38 infants at 4-month intervals from birth until 2 years of age. Nineteen of the infants developed one episode of HRV diarrhea each, and they were matched by age and birth weight with the other 19 infants, who did not develop HRV diarrhea during the follow-up period. We estimated the incidence rate of HRV infection for the two groups of infants combined to be a minimum of 1.34 episodes per infant per year, which is 22 times more common than the occurrence of overt disease caused by the virus in this community. The infection occurred constantly throughout the first 2 years of infancy, whereas all but one of the 19 episodes of overt disease occurred before 12 months of age. Seven of these overt episodes were preceded by at least one episode of subclinical infection earlier, and the other seven were probably due to primary HRV infection. The remaining five episodes occurred before 4 months of age, so that we could not ascertain whether they were due to primary infections because of the presence of maternal antibodies. We showed that levels of HRV antibodies in serum specimens obtained before clinical onset of diarrhea varied widely, and, for most infants in the diarrheal group, levels of these antibodies were similar to those in the serum specimens obtained at the same times from the corresponding age- and birth weight-matched control infants. Nevertheless, the age at which overt disease caused by HRV was most prevalent coincided with the time when the maternal antibodies had declined to low levels but the infants had not yet acquired high titers of these antibodies in their sera.