Browsing by Author "Lei, Shaohua"

Now showing 1 - 13 of 13
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    Cell-free protein synthesis of norovirus virus-like particles
    Sheng, Jiayuan; Lei, Shaohua; Yuan, Lijuan; Feng, Xueyang (Royal Society of Chemistry, 2017-05-25)
    Norovirus vaccine development largely depends on recombinant virus-like-particles (VLPs). Norovirus VLPs have been produced in several cell-based expression systems with long production times. Here we report, for the first time, that norovirus VLPs can be expressed and assembled by using a cell-free protein expression system within four hours.
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    Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs
    Lei, Shaohua; Samuel, Helen; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Wen, Ke; Weiss, Mariah; Li, Guohua; Yang, Xingdong; Jiang, Xi; Yuan, Lijuan (2016-04-26)
    Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 10(4) genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization.
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    Evaluation of the 50% Infectious Dose of Human Norovirus Cin-2 in Gnotobiotic Pigs: A Comparison of Classical and Contemporary Methods for Endpoint Estimation
    Ramesh, Ashwin; Parreño, Viviana; Schmidt, Philip J.; Lei, Shaohua; Zhong, Weiming; Jiang, Xi; Emelko, Monica B.; Yuan, Lijuan (MDPI, 2020-08-28)
    Human noroviruses (HuNoVs) are the leading causative agents of epidemic and sporadic acute gastroenteritis that affect people of all ages worldwide. However, very few dose–response studies have been carried out to determine the median infectious dose of HuNoVs. In this study, we evaluated the median infectious dose (ID50) and diarrhea dose (DD50) of the GII.4/2003 variant of HuNoV (Cin-2) in the gnotobiotic pig model of HuNoV infection and disease. Using various mathematical approaches (Reed–Muench, Dragstedt–Behrens, Spearman–Karber, exponential, approximate beta-Poisson dose–response models, and area under the curve methods), we estimated the ID50 and DD50 to be between 2400–3400 RNA copies, and 21,000–38,000 RNA copies, respectively. Contemporary dose–response models offer greater flexibility and accuracy in estimating ID50. In contrast to classical methods of endpoint estimation, dose–response modelling allows seamless analyses of data that may include inconsistent dilution factors between doses or numbers of subjects per dose group, or small numbers of subjects. Although this investigation is consistent with state-of-the-art ID50 determinations and offers an advancement in clinical data analysis, it is important to underscore that such analyses remain confounded by pathogen aggregation. Regardless, challenging virus strain ID50 determination is crucial for identifying the true infectiousness of HuNoVs and for the accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model.
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    High Protective Efficacy of Probiotics and Rice Bran against Human Norovirus Infection and Diarrhea in Gnotobiotic Pigs
    Lei, Shaohua; Ramesh, Ashwin; Twitchell, Erica; Wen, Ke; Bui, Tammy; Weiss, Mariah; Yang, Xingdong; Kocher, Jacob; Li, Guohua; Giri-Rachman, Ernawati; Trang, Nguyen Van; Jiang, Xi; Ryan, Elizabeth P.; Yuan, Lijuan (2016)
    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.
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    High protective efficacy of rice bran against human rotavirus diarrhea via enhancing probiotic growth, gut barrier function, and innate immunity
    Yang, Xingdong; Twitchell, Erica; Li, Guohua; Wen, Ke; Weiss, Mariah; Kocher, Jacob; Lei, Shaohua; Ramesh, Ashwin; Ryan, Elizabeth P.; Yuan, Lijuan (Nature Publishing Group, 2015-10-13)
    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-formingunits 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.
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    Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency
    Lei, Shaohua; Ryu, Junghyun; Wen, Ke; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Weiss, Mariah; Li, Guohua; Samuel, Helen; Clark-Deener, Sherrie; Jiang, Xi; Lee, Kiho; Yuan, Lijuan (Nature Publishing Group, 2016-04-27)
    Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.
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    Modeling human enteric dysbiosis and rotavirus immunity in gnotobiotic pigs
    Twitchell, Erica; Tin, Christine; Wen, Ke; Zhang, Husen; Becker-Dreps, Sylvia; Azcarate-Peril, M. Andrea; Vilchez, Samuel; Li, Guohua; Ramesh, Ashwin; Weiss, Mariah; Lei, Shaohua; Bui, Tammy; Yang, Xingdong; Schultz-Cherry, Stacey L.; Yuan, Lijuan (2016)
    BACKGROUND: Rotavirus vaccines have poor efficacy in infants from low- and middle-income countries. Gut microbiota is thought to influence the immune response to oral vaccines. Thus, we developed a gnotobiotic (Gn) pig model of enteric dysbiosis to study the effects of human gut microbiota (HGM) on immune responses to rotavirus vaccination, and the effects of rotavirus challenge on the HGM by colonizing Gn pigs with healthy HGM (HHGM) or unhealthy HGM (UHGM). The UHGM was from a Nicaraguan infant with a high enteropathy score (ES) and no seroconversion following administration of oral rotavirus vaccine, while the converse was characteristic of the HHGM. Pigs were vaccinated, a subset was challenged, and immune responses and gut microbiota were evaluated. RESULTS: Significantly more rotavirus-specific IFN-γ producing T cells were in the ileum, spleen, and blood of HHGM than those in UHGM pigs after three vaccine doses, suggesting HHGM induces stronger cell-mediated immunity than UHGM. There were significant correlations between multiple Operational Taxonomic Units (OTUs) and frequencies of IFN-γ producing T cells at the time of challenge. There were significant positive correlations between Collinsella and CD8+ T cells in blood and ileum, as well as CD4+ T cells in blood, whereas significant negative correlations between Clostridium and Anaerococcus, and ileal CD8+ and CD4+ T cells. Differences in alpha diversity and relative abundances of OTUs were detected between the groups both before and after rotavirus challenge. CONCLUSION: Alterations in microbiome diversity and composition along with correlations between certain microbial taxa and T cell responses warrant further investigation into the role of the gut microbiota and certain microbial species on enteric immunity. Our results support the use of HGM transplanted Gn pigs as a model of human dysbiosis during enteric infection, and oral vaccine responses.
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    Modeling human enteric dysbiosis and rotavirus immunity in gnotobiotic pigs. [poster]
    Twitchell, Erica; Tin, Christine; Wen, Ke; Zhang, Husen; Becker-Dreps, Sylvia; Azcarate-Peril, M. Andrea; Vilchez, Samuel; Li, Guohua; Ramesh, Ashwin; Weiss, Mariah; Lei, Shaohua; Bui, Tammy; Yang, Xingdong; Schultz-Cherry, Stacey L.; Yuan, Lijuan (2016-12)
    Background Oral vaccines, such as those for rotavirus are less efficacious in children from underdeveloped regions, where most severe disease occurs, than in children from more affluent areas. This disparity may be due to altered gut microbiota composition (dysbiosis), environmental enteropathy (EE), high maternal antibody titers, malnutrition, or influence of concurrent enteropathogens. Composition of gut microbiota in children is influenced by method of delivery, environmental hygiene and nutritional status. Studies have shown composition of gut microbiota to be significantly different between African and northern European infants and between malnourished and well-nourished children. A recent study has shown that EE was associated with failure of the oral rotavirus vaccine Rotarix, and underperformance of the oral polio vaccine. An animal model to study the effects of enteric dysbiosis on oral vaccine immunity is needed to evaluate potential treatments to reverse the dysbiosis and/or improve vaccine efficacy. Pigs and humans have similar immune systems, high genomic and protein sequence homology, omnivorous diet, and colonic fermentation, making pigs valuable models in biomedical research. The neonatal gnotobiotic (Gn) pig is a well-established model of human rotavirus disease and immunity.
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    A New Gnotobiotic Pig Model of P[6] Human Rotavirus Infection and Disease for Preclinical Evaluation of Rotavirus Vaccines
    Nyblade, Charlotte; Hensley, Casey; Parreño, Viviana; Zhou, Peng; Frazier, Maggie; Frazier, Annie; Ramesh, Ashwin; Lei, Shaohua; Degiuseppe, Juan Ignacio; Tan, Ming; Yuan, Lijuan (MDPI, 2022-12-15)
    Human rotavirus (HRV) is a leading cause of gastroenteritis in children under 5 years of age. Licensed vaccines containing G1P[8] and G1-4P[8] strains are less efficacious against newly emerging P[6] strains, indicating an urgent need for better cross protective vaccines. Here, we report our development of a new gnotobiotic (Gn) pig model of P[6] HRV infection and disease as a tool for evaluating potential vaccine candidates. The Arg HRV (G4P[6]) strain was derived from a diarrheic human infant stool sample and determined to be free of other viruses by metagenomic sequencing. Neonatal Gn pigs were orally inoculated with the stool suspension containing 5.6 × 105 fluorescent focus units (FFU) of the virus. Small and large intestinal contents were collected at post inoculation day 2 or 3. The virus was passaged 6 times in neonatal Gn pigs to generate a large inoculum pool. Next, 33–34 day old Gn pigs were orally inoculated with 10−2, 103, 104, and 105 FFU of Arg HRV to determine the optimal challenge dose. All pigs developed clinical signs of infection, regardless of the inoculum dose. The optimal challenge dose was determined to be 105 FFU. This new Gn pig model is ready to be used to assess the protective efficacy of candidate monovalent and multivalent vaccines against P[6] HRV.
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    Parenterally Administered P24-VP8* Nanoparticle Vaccine Conferred Strong Protection against Rotavirus Diarrhea and Virus Shedding in Gnotobiotic Pigs
    Ramesh, Ashwin; Mao, Jiangdi; Lei, Shaohua; Twitchell, Erica; Shiraz, Ashton K.; Jiang, Xi; Tan, Ming; Yuan, Lijuan (MDPI, 2019-11-06)
    Current live rotavirus vaccines are costly with increased risk of intussusception due to vaccine replication in the gut of vaccinated children. New vaccines with improved safety and cost-effectiveness are needed. In this study, we assessed the immunogenicity and protective efficacy of a novel P24-VP8* nanoparticle vaccine using the gnotobiotic (Gn) pig model of human rotavirus infection and disease. Three doses of P24-VP8* (200 μg/dose) intramuscular vaccine with Al(OH)3 adjuvant (600 μg) conferred significant protection against infection and diarrhea after challenge with virulent Wa strain rotavirus. This was indicated by the significant reduction in the mean duration of diarrhea, virus shedding in feces, and significantly lower fecal cumulative consistency scores in post-challenge day (PCD) 1–7 among vaccinated pigs compared to the mock immunized controls. The P24-VP8* vaccine was highly immunogenic in Gn pigs. It induced strong VP8*-specific serum IgG and Wa-specific virus-neutralizing antibody responses from post-inoculation day 21 to PCD 7, but did not induce serum or intestinal IgA antibody responses or a strong effector T cell response, which are consistent with the immunization route, the adjuvant used, and the nature of the non-replicating vaccine. The findings are highly translatable and thus will facilitate clinical trials of the P24-VP8* nanoparticle vaccine.
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    Pathogenesis, immunity, and prevention of human norovirus infection in gnotobiotic pigs
    Lei, Shaohua (Virginia Tech, 2018-04-23)
    Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis and responsible for the deaths of over 200,000 children each year worldwide. HuNoV research has been hampered by the long absence of a readily reproducible cell culture system and a suitable small animal model, while gnotobiotic (Gn) pigs have been a unique animal model for understanding HuNoV pathogenesis and immunity, as well as evaluating vaccine and therapeutics. Recent reports of HuNoVs infection and replication in B cells supplemented with commensal bacteria Enterobacter cloacae and in Blab/c mice deficient in RAG/IL2RG have gained extensive attention, and my studies utilized the well-established Gn pig model to investigate the effects of these two interventions on HuNoV infection. Surprisingly, the colonization of E. cloacae inhibited HuNoV infectivity in Gn pigs, evidenced by the significantly reduced HuNoV shedding in feces and HuNoV titers in intestinal tissues and blood compared to control pigs. Moreover, HuNoV infection of enterocytes but not B cells was observed with or without E. cloacae colonization, indicating B cells were not a target cell type for HuNoV in Gn pigs. On the other hand, using RAG2/IL2RG deficient pigs generated by CRISPR/Cas9 system, with confirmed severe combined immunodeficiency, I evaluated the effects of host immune responses on HuNoV infection. Compared to wild-type Gn pigs, longer HuNoV shedding was observed in RAG2/IL2RG deficient pigs (16 versus 27 days), and higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs. In addition, I evaluated dietary interventions including probiotics and rice bran using Gn pig model of HuNoV infection and diarrhea. While the colonization of probiotic bacteria Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) in Gn pigs completely inhibited HuNoV fecal shedding, the two cocktail regimens, in which rice bran feeding started either 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized Gn pigs, exhibited dramatic anti-HuNoV effects, including reduced incidence and shorter duration of diarrhea, as well as shorter duration of virus fecal shedding. The anti-HuNoV effects of the cocktail regimens were associated with the enhanced IFN-𝛾⁺ T cell responses, increased production of intestinal IgA and IgG, and longer villus length. Taken together, my dissertation work improves our understanding of HuNoV infection and immunity, and further supports for Gn pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions.
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    A RNA Virus Reference Database (RVRD) to Enhance Virus Detection in Metagenomic Data
    Lei, Shaohua (Virginia Tech, 2018-10-16)
    With the great promise that metagenomics holds in exploring virome composition and discovering novel virus species, there is a pressing demand for comprehensive and up-to-date reference databases to enhance the downstream bioinformatics analysis. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation of RNA virus genomes downloaded from the three major virus sequence databases including NCBI, ViralZone, and ViPR. To reduce viral sequence redundancy caused by multiple identical or nearly identical sequences, sequences were first clustered and all sequences except one in a cluster that have more than 98% identity to one another were removed. Other identity cutoffs were also examined, and Hepatitis C virus genomes were studied in detail as an example. Using the 98% identity cutoff, sequences obtained from ViPR were combined with the unique RNA virus references from NCBI and ViralZone to generate the final RVRD. The resulting RVRD contained 23,085 sequences, nearly 5 times the size of NCBI RNA virus reference, and had a broad coverage of RNA virus families, with significant expansion on circular ssRNA virus and pathogenic virus families. Compared to NCBI RNA virus reference in performance evaluation, using RVRD as reference database identified more RNA virus species in RNAseq data derived from wastewater samples. Moreover, using RVRD as reference database also led to the discovery of porcine rotavirus as the etiology of unexplained diarrhea observed in pigs. RVRD is publicly available for enhancing RNA virus metagenomics.
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    Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs
    Kocher, Jacob; Castellucci, Tammy Bui; Wen, Ke; Li, Guohua; Yang, Xingdong; Lei, Shaohua; Jiang, Xi; Yuan, Lijuan (MDPI, 2021-07-01)
    Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.