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

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

Abstracts Background Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. Methods First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. Results Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. Conclusions Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research

Clinical signs and rotavirus fecal shedding in AttHRV-vaccinated HGM pigs after VirHRV challenge.

a<p>Pigs with daily fecal scores of ≥2 were considered diarrheic. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid.</p>b<p>For durations of diarrhea and virus shedding, if no diarrhea or virus shedding until the euthanasia day (PCD7), the duration (days) were recorded as 0 and the onset (days) were as 8 for statistical analysis.</p>c<p>Mean cumulative score calculation included all the pigs in each group.</p>d<p>Standard error of the mean.</p>e<p>FFU, fluorescent focus forming units. Geometric mean peak titers were calculated among pigs that shed virus.</p><p>*Fisher's exact test or ** Kruskal-Wallis rank sum test was used for comparisons. Different letters indicate significant differences among treatment groups (p<0.05), while shared letters indicate no significant difference.</p

Rotavirus-specific IFN-γ producing T cell responses in HGM transplanted Gn pigs fed with different doses of LGG.

<p>Data are presented as mean frequency ± standard error of the mean (n = 4–6). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone-0094504-g002" target="_blank">Fig. 2</a> legend for detection of rotavirus-specific IFN-γ producing T cell and statistical analysis.</p

T cell responses in AttHRV vaccinated pigs with or without HGM transplantation.

<p>MNCs were stimulated with semi-purified AttHRV antigen <i>in vitro</i> for 17 hrs. Brefeldin A was added for the last 5 hrs to block secretion of cytokines produced by the T cells. HRV-specific IFN-γ producing CD4+ and CD8+ T cells was detected by intracellular staining and flow cytometry as we previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone.0094504-Yuan5" target="_blank">[16]</a>. The frequencies of IFN−γ+CD4+/CD8+ T cells were expressed as percentages among total CD3+ T cells (A and B, middle panel). All mean frequencies are reported after subtraction of the background frequencies. The absolute numbers of CD3+CD4+/CD8+ cells and IFN−γ+CD3+CD4+/CD8+ cells per tissue (A and B, top and bottom panels) were calculated based on the frequencies of CD3+CD4+/CD8+ cells and IFN−γ+CD3+CD4+/CD8+ cells, respectively, and the total number of MNCs isolated from each tissue. Data are presented as mean number or frequency ± standard error of the mean (n = 4–12). The sign “*” indicates the significant difference between groups (Kruskal–Wallis test, p<0.05).</p

Number of pigs in non-HGM group and HGM groups at beginning of study and at euthanasia on PID 28 and PCD 7.

<p>Number of pigs in non-HGM group and HGM groups at beginning of study and at euthanasia on PID 28 and PCD 7.</p

Rotavirus-specific serum IgA and IgG antibody responses (A and B) and rotavirus-specific IgA antibody responses in small intestine contents (C) of Gn pigs transplanted with HGM and fed different doses of LGG.

<p>Rotavirus-specific antibody titers were measured by an indirect isotype-specific antibody ELISA and presented as geometric mean titers for each treatment group + standard error of the mean (n = 3–10 for serum samples and n = 3–6 for small intestine content samples). Different capital letters (A, B, or C) indicate significant differences in antibody titers compared among different groups at the same time points; different lower case letters (a, b, c, d) indicate significant difference between different time points in the same group (Kruskal–Wallis test, p<0.05), whereas shared letters indicate no significant difference. The sign “*” indicates significant differences in IgA titers in small intestine contents between groups at the same time points and the symbol “Δ” indicates significant increases in IgA titers at PCD7 compared to PID28 for the same group (Kruskal–Wallis test, p<0.05).</p

Clinical sign and virus shedding in AttHRV vaccinated pigs with or without HGM transplantation (A) and LGG shedding in fecal samples and large intestinal contents of HGM transplanted Gn pigs fed with or without LGG (B).

<p>After VirHRV challenge, pigs were monitored for 7 days for incidence of diarrhea, fecal score and virus shedding. Data are presented as mean ± standard error of the mean (n = 12 for AttHRV group; n = 4 for HGM+AttHRV group). The sign “*” in (A) indicates significant difference between groups (Kruskal–Wallis test, <i>p</i><0.05). LGG amounts at different time points were determined by strain-specific real-time PCR and are presented as mean counts/ml ± standard error of the mean (n = 7–10 for fecal samples and n = 3–6 for large intestinal content samples). The sign “*” in (B) indicates significant differences between groups at the same time points and the symbol “Δ” indicates significant increases in LGG numbers compared to PID 5 for the same group (Kruskal–Wallis test, p<0.05).</p

Treg responses in AttHRV vaccinated pigs with or without HGM transplantation.

<p>MNCs were stained freshly without <i>in vitro</i> stimulation. The frequencies of Tregs were expressed as the percentages among gated MNCs (A, top panel). The absolute numbers of Tregs per tissue were calculated based on the frequencies of Tregs and the total number of MNCs isolated from each tissue (A, bottom panel). The frequencies of IL−10+ or TGF−β+ Tregs were expressed as the percentages of IL−10+ or TGF−β+ cells among the Tregs (B). Data are presented as mean number or frequency ± standard error of the mean (n = 4–9). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone-0094504-g002" target="_blank">Fig. 2</a> legend for statistical analysis.</p

Additional file 1: Table S1. of Lactobacillus rhamnosus GG modulates innate signaling pathway and cytokine responses to rotavirus vaccine in intestinal mononuclear cells of gnotobiotic pigs transplanted with human gut microbiota

The primers used in this study. (PDF 54 kb