ABSTRACT
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.
ABSTRACT
Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.
Subject(s)
Rotavirus Infections , Rotavirus , Child , Animals , Humans , Swine , Mice , Lymphoid Tissue , Proteins , Immunoglobulin M , Immunity , Germ-Free Life , Salivary GlandsABSTRACT
Human rotavirus (HRV) is the causative agent of severe dehydrating diarrhea in children under the age of five, resulting in up to 215,000 deaths each year. These deaths almost exclusively occur in low- and middle-income countries where vaccine efficacy is the lowest due to chronic malnutrition, gut dysbiosis, and concurrent enteric viral infection. Parenteral vaccines for HRV are particularly attractive as they avoid many of the concerns associated with currently used live oral vaccines. In this study, a two-dose intramuscular (IM) regimen of the trivalent, nanoparticle-based, nonreplicating HRV vaccine (trivalent S60-VP8*), utilizing the shell (S) domain of the capsid of norovirus as an HRV VP8* antigen display platform, was evaluated for immunogenicity and protective efficacy against P[6] and P[8] HRV using gnotobiotic pig models. A prime-boost strategy using one dose of the oral Rotarix® vaccine, followed by one dose of the IM trivalent nanoparticle vaccine was also evaluated. Both regimens were highly immunogenic in inducing serum virus neutralizing, IgG, and IgA antibodies. The two vaccine regimens failed to confer significant protection against diarrhea; however, the prime-boost regimen significantly shortened the duration of virus shedding in pigs challenged orally with the virulent Wa (G1P[8]) HRV and significantly shortened the mean duration of virus shedding, mean peak titer, and area under the curve of virus shedding after challenge with Arg (G4P[6]) HRV. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG antibody-secreting cells (ASCs) in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6]- and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. These results suggest the promise of and warrant further investigation into the oral priming and parenteral boosting strategy for future HRV vaccines.
ABSTRACT
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.
Subject(s)
Rotavirus Infections , Rotavirus Vaccines , Rotavirus , Infant , Child , Infant, Newborn , Humans , Animals , Swine , Child, Preschool , Rotavirus Infections/prevention & control , Rotavirus Infections/veterinary , Feces , Germ-Free LifeABSTRACT
Clostridioides difficile (C. difficile) is a gram-positive, spore-forming, anaerobic bacterium known to be the most common cause of hospital-acquired and antibiotic-associated diarrhea. C. difficile infection rates are on the rise worldwide and treatment options are limited, indicating a clear need for novel therapeutics. Gnotobiotic piglets are an excellent model to reproduce the acute pseudomembranous colitis (PMC) caused by C. difficile due to their physiological similarities to humans and high susceptibility to infection. Here, we established a gnotobiotic pig model of C. difficile infection and disease using a hypervirulent strain. C. difficile-infected pigs displayed classic signs of C. difficile infection, including severe diarrhea and weight loss. Inoculated pigs had severe gross and microscopic intestinal lesions. C. difficile infection caused an increase in pro-inflammatory cytokines in samples of serum, large intestinal contents, and pleural effusion. C. difficile spores and toxins were detected in the feces of inoculated animals as tested by anaerobic culture and cytotoxicity assays. Successful establishment of this model is key for future work as therapeutics can be evaluated in an environment that accurately mimics what happens in humans. The model is especially suitable for evaluating potential prophylactics and therapeutics, including vaccines and passive immune strategies.
ABSTRACT
PURPOSE: The purpose of the study was to determine whether a model-based iterative reconstruction (MBIR) technique improves diagnostic confidence and detection of pulmonary embolism (PE) compared with hybrid iterative reconstruction (HIR) and filtered back projection (FBP) reconstructions in patients undergoing computed tomography pulmonary angiography. MATERIALS AND METHODS: The study was approved by our institutional review board. Fifty patients underwent computed tomography pulmonary angiography at 100 kV using standard departmental protocols. Twenty-two of 50 patients had studies positive for PE. All 50 studies were reconstructed using FBP, HIR, and MBIR. After image randomization, 5 thoracic radiologists and 2 thoracic radiology fellows graded each study on a scale of 1 (very poor) to 5 (ideal) in 4 subjective categories: diagnostic confidence, noise, pulmonary artery enhancement, and plastic appearance. Readers assessed each study for the presence of PE. Parametric and nonparametric data were analyzed with repeated measures and Friedman analysis of variance, respectively. RESULTS: For the 154 positive studies (7 readers × 22 positive studies), pooled sensitivity for detection of PE was 76% (117/154), 78.6% (121/154), and 82.5% (127/154) using FBP, HIR, and MBIR, respectively. PE detection was significantly higher using MBIR compared with FBP (P = 0.016) and HIR (P = 0.046). Because of nonsignificant increase in FP studies using HIR and MBIR, accuracy with MBIR (88.6%), HIR (87.1%), and FBP (87.7%) was similar. Compared with FBP, MBIR led to a significant subjective increase in diagnostic confidence, noise, and enhancement in 6/7, 6/7, and 7/7 readers, respectively. Compared with HIR, MBIR led to significant subjective increase in diagnostic confidence, noise, and enhancement in 5/7, 5/7, and 7/7 readers, respectively. MBIR led to a subjective increase in plastic appearance in all 7 readers compared with both FBP and HIR. CONCLUSIONS: MBIR led to significant increase in PE detection compared with FBP and HIR. MBIR led to qualitative improvements in diagnostic confidence, perceived noise, and perceived enhancement compared with FBP and HIR.