Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

OBJECTIVE: New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. METHODS: Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. RESULTS: Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. CONCLUSION: This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Ultrasound-guided noninvasive pancreas ablation using histotripsy: feasibility study in an in vivo porcine model.

Pancreatic cancer is a malignant disease associated with poor survival and nearly 80% present with unresectable tumors. Treatments such as chemotherapy and radiation therapy have shown overall improved survival benefits, albeit limited. Histotripsy is a noninvasive, non-ionizing, and non-thermal focused ultrasound ablation modality that has shown efficacy in treating hepatic tumors and other malignancies. In this novel study, we investigate histotripsy for noninvasive pancreas ablation in a pig model. In two studies, histotripsy was applied to the healthy pancreas in 11 pigs using a custom 32-element, 500 kHz histotripsy transducer attached to a clinical histotripsy system, with treatments guided by real-time ultrasound imaging. A pilot study was conducted in 3 fasted pigs with histotripsy applied at a pulse repetition frequency (PRF) of 500 Hz. Results showed no pancreas visualization on coaxial ultrasound imaging due to overlying intestinal gas, resulting in off-target injury and no pancreas damage. To minimize gas, a second group of pigs (n = 8) were fed a custard diet containing simethicone and bisacodyl. Pigs were euthanized immediately (n = 4) or survived for 1 week (n = 4) post-treatment. Damage to the pancreas and surrounding tissue was characterized using gross morphology, histological analysis, and CT imaging. Results showed histotripsy bubble clouds were generated inside pancreases that were visually maintained on coaxial ultrasound (n = 4), with 2 pigs exhibiting off-target damage. For chronic animals, results showed the treatments were well-tolerated with no complication signs or changes in blood markers. This study provides initial evidence suggesting histotripsy's potential for noninvasive pancreas ablation and warrants further evaluation in more comprehensive studies.

Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study.

Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.

Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood-brain barrier, and invades the central nervous system.

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain-Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood-brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

In-plane and out-of-plane deformations of gilt utero-sacral ligaments.

The uterosacral ligaments (USLs) are supportive structures of the uterus and apical vagina. The mechanical function of these ligaments within the pelvic floor is crucial not only in normal physiological conditions but also in reconstructive surgeries for pelvic organ prolapse. Discrepancies in their anatomical and histological description exist in the literature, but such discrepancies are likely due to large variations of these structures. This makes mechanical testing very challenging, requiring the development of advanced methods for characterizing their mechanical properties. This study proposes the use of planar biaxial testing, digital image correlation (DIC), and optical coherence tomography (OCT) to quantify the deformations of the USLs, both in-plane and out-of-plane. Using the gilts as an animal model, the USLs were found to deform significantly less in their main direction (MD) of in vivo loading than in the direction perpendicular to it (PD) at increasing equibiaxial stresses. Under constant equibiaxial loading, the USLs deform over time equally, at comparable rates in both the MD and PD. The thickness of the USLs decreases as the equibiaxial loading increases but, under constant equibiaxial loading, the thickness increases in some specimens and decreases in others. These findings could contribute to the design of new mesh materials that augment the support function of USLs as well as noninvasive diagnostic tools for evaluating the integrity of the USLs.

Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

Quantification of zearalenone and α-zearalenol in swine liver and reproductive tissues using GC-MS.

The mycotoxin zearalenone (ZEN) is a common contaminant of swine feed which has been related to a wide range of reproductive anomalies in swine, such as pelvic organ prolapse, anestrous, and pseudopregnancy. New information is needed to understand how ZEN and related metabolites accumulate in swine reproductive tissues. We conducted a feeding study to track ZEN and the metabolite α-zearalenol (α-ZEL) in swine liver and reproductive tissues. Thirty pubertal gilts were randomly assigned one of three treatments, with ten pigs in each treatment group: (1) base feed with solvent for 21 days, (2) ZEN-spiked feed for seven days followed by base feed with solvent for 14 days, and (3) ZEN-spiked feed for 21 days. At the end of the trial, liver, anterior vagina, posterior vagina, cervix, uterus, ovaries, and broad ligament were collected from pigs. ZEN was found in the anterior vagina, posterior vagina, cervix, and ovaries, with significantly higher concentrations in the cervix relative to other reproductive tissues. ZEN and α-ZEL were found in liver tissue from pigs in each treatment group. Our results show that ZEN accumulates more in the cervix than other reproductive tissues. The presence of ZEN in reproductive tissues may be indicative of ZEN-related reproductive symptoms. Future work could examine how ZEN concentrations vary in reproductive tissues as a factor of the pigs age, weight, sex, or parity, to establish parameters that make pig more sensitive to ZEN.

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity.

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope 748YSNIGVCK755 from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).

Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos.

BACKGROUND: The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. RESULTS: First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5' side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5' side, indicating unpredictability of the events. CONCLUSIONS: The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH-/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH-/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH-/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH-/- knockout pigs, suggesting that the Ig heavy chain JH-/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH-/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH-/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH-/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH-/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Supplementing Ca salts of soybean oil after artificial insemination increases pregnancy success in Bos taurus beef cows.

Two experiments investigated the effects of supplementing Ca salts of soybean oil (CSSO) during early gestation on reproductive function and pregnancy rates to AI in Bos taurus beef cows. In Exp. 1, 771 suckled, lactating, multiparous Angus cows were divided into 22 groups of approximately 35 cows per group and timed inseminated on day 0. After AI, groups were assigned randomly to receive (as-fed basis) 100 g of ground corn + 100 g of soybean meal per cow/d, in addition to 1) 100 g/cow daily of CSSO (n = 11) or 2) 87 g of prilled saturated fat + 13 g of limestone per cow/d (CON; n = 11). Groups were maintained in individual tall fescue-dominated pastures and offered treatments from day 0 to 21. Pregnancy status was determined between days 45 and 55 via transrectal ultrasonography. Cows receiving CSSO had greater (P = 0.01) pregnancy rates to timed AI compared with CON (60.2 vs. 51.7%; SEM = 4.2). In Exp. 2, 90 suckled, lactating, multiparous Angus × Hereford cows housed in 18 drylot pens (5 cows per pen) were assigned to the same timed AI program and treatments from Exp. 1 (9 pens per treatment) and received 20 kg/d (DM basis) of grass-alfalfa hay. Transrectal ultrasonography was performed to verify ovulation and corpus luteum (CL) volume before AI (day 0), on days 7 and 15. After ultrasonography on day 15, cows diagnosed without a CL on day 0, but with a CL greater than 0.38 cm3 in volume on days 7 and 15 (2 or 3 cows per pen; CSSO, n = 20; CON, n = 24), were assigned to conceptus collection via transcervical flushing and endometrial biopsy in the uterine horn ipsilateral to the CL. Blood samples were collected for FA analysis on days 0, 7, and 15. Blood was collected from cows not assigned to conceptus collection for whole-blood RNA extraction on day 20 and for pregnancy diagnosis on day 30 by measuring concentrations of pregnancy-associated glycoproteins. Cows receiving CSSO had greater (P ≤ 0.04) mean plasma concentrations of linoleic acid and ω-6 FA compared with CON on days 7 and 15. Moreover, CSSO supplementation increased (P = 0.05) mRNA expression of interferon-tau by the conceptus and blood mRNA expression of interferon-stimulated gene 15 and 20,50-oligoadenylate synthetase on day 20 in gestating cows. Hence, post-AI CSSO supplementation to B. taurus beef cows improved pregnancy rates to timed AI, which can be associated with increased mRNA expression of interferon-tau by the conceptus when CSSO is supplemented during early gestation.

Pig model mimicking chronic hepatitis E virus infection in immunocompromised patients to assess immune correlates during chronicity.

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ-specific CD4+ T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α-specific CD8+ T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs.

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.

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.

BACKGROUND: A better understanding of mechanisms underlying dose-effects of probiotics in their applications as treatments of intestinal infectious or inflammatory diseases and as vaccine adjuvant is needed. In this study, we evaluated the modulatory effects of Lactobacillus rhamnosus GG (LGG) on transplanted human gut microbiota (HGM) and on small intestinal immune cell signaling pathways in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV) vaccine. RESULTS: Neonatal HGM transplanted pigs were given two doses of AttHRV on 5 and 15 days of age and were divided into three groups: none-LGG (AttHRV), 9-doses LGG (AttHRV + LGG9X), and 14-doses LGG (AttHRV + LGG14X) (n = 3-4). At post-AttHRV-inoculation day 28, all pigs were euthanized and intestinal contents and ileal tissue and mononuclear cells (MNC) were collected. AttHRV + LGG14X pigs had significantly increased LGG titers in the large intestinal contents and shifted structure of the microbiota as indicated by the formation of a cluster that is separated from the cluster formed by the AttHRV and AttHRV + LGG9X pigs. The increase in LGG titers concurred with significantly increased ileal HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine reported in our previous publication, suggesting pro-Th1 adjuvant effects of the LGG. Both 9- and 14-doses LGG fed pig groups had significantly higher IkBα level and p-p38/p38 ratio, while significantly lower p-ERK/ERK ratio than the AttHRV pigs, suggesting activation of regulatory signals during immune activation. However, 9-doses, but not 14-doses LGG fed pigs had enhanced IL-6, IL-10, TNF-α, TLR9 mRNA levels, and p38 MAPK and ERK expressions in ileal MNC. Increased TLR9 mRNA was in parallel with higher mRNA levels of cytokines, p-NF-kB and higher p-p38/p38 ratio in MNC of the AttHRV + LGG9X pigs. CONCLUSIONS: The relationship between modulation of gut microbiota and regulation of host immunity by different doses of probiotics is complex. LGG exerted divergent dose-dependent effects on the intestinal immune cell signaling pathway responses, with 9-doses LGG being more effective in activating the innate immunostimulating TLR9 signaling pathway than 14-doses in the HGM pigs vaccinated with AttHRV.

Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency.

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.

B-Cell-Deficient and CD8 T-Cell-Depleted Gnotobiotic Pigs for the Study of Human Rotavirus Vaccine-Induced Protective Immune Responses.

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(-) γδ 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.

Lactobacillus rhamnosus GG Dosage Affects the Adjuvanticity and Protection Against Rotavirus Diarrhea in Gnotobiotic Pigs.

OBJECTIVES: The use of immunostimulatory strains of probiotics as adjuvants has been increasingly recognized as a promising approach in enhancing vaccine immunogenicity; however, dose effects of probiotic adjuvants are not well defined. In the present study, we examined dose effects of a commonly used probiotic strain, Lactobacillus rhamnosus GG (LGG), on immunomodulation with 2 different dosages. METHODS: Neonatal gnotobiotic pigs were inoculated with 2 oral doses of attenuated human rotavirus (AttHRV) vaccines and fed with 5 doses (LGG5X; total 2.1 × 10(6) colony-forming units) or 9 doses (LGG9X; total 3.2 × 10(6) colony-forming units) of LGG, starting at 3 days of age. RESULTS: Both LGG feeding regimens enhanced the protection rate of AttHRV vaccine against diarrhea on virulent human rotavirus challenge. LGG5X, but not LGG9X, significantly enhanced rotavirus-specific intestinal memory B-cell responses to AttHRV; LGG5X also significantly enhanced virus-specific intestinal immunoglobulin A (IgA) antibody-secreting cell responses. Both regimens significantly enhanced rotavirus-specific serum IgA antibody responses to AttHRV. They also enhanced rotavirus-specific interferon-γ-producing effector/memory T-cell responses to AttHRV vaccine, with LGG9X being more effective than LGG5X, and both regimens downregulated CD4+CD25-FoxP3+ regulatory T (Treg) cell responses in most lymphoid tissues examined prechallenge and postchallenge and maintained the CD4+CD25+FoxP3+ Treg population in the ileum and intraepithelial lymphocyte postchallenge. LGG9X, however, did not significantly reduce total CD4+CD25-FoxP3+ Treg frequencies in the intestine and transforming growth factor-ß-producing and interleukin (IL)-10-producing Treg frequencies in the blood. CONCLUSIONS: These results indicate that LGG at both dosages functioned as effective probiotic adjuvant for AttHRV vaccine, but different dosages differentially modulated immune responses to favor either the mucosal IgA response (LGG5X) or the T-cell response (LGG9X).

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.

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 × 10(6) colony-forming units [CFU]), intermediate-dose LA (MidLA) (9 doses, total 3.2 × 10(9) CFU), low-dose LA (LoLA) (5 doses, total 2.1 × 10(6) 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.