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.

Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs.

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.

Combined Live Oral Priming and Intramuscular Boosting Regimen with Rotarix® and a Nanoparticle-Based Trivalent Rotavirus Vaccine Evaluated in Gnotobiotic Pig Models of G4P[6] and G1P[8] Human Rotavirus Infection.

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.

A New Gnotobiotic Pig Model of P[6] Human Rotavirus Infection and Disease for Preclinical Evaluation of Rotavirus Vaccines.

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.

Establishment of a gnotobiotic pig model of Clostridioides difficile infection and disease.

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.

Thermostable, Dissolvable Buccal Film Rotavirus Vaccine Is Highly Effective in Neonatal Gnotobiotic Pig Challenge Model.

Difficulties related to storage and transport of currently available live oral rotavirus vaccines can have detrimental consequences on the efficacy of the vaccines. Thus, there is a great need for thermostable vaccines that can eliminate the necessity for cold chain storage or reconstitution before administration. In this study, we developed a dissolvable oral polymeric film comprised of a live attenuated thermostable tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) powder and antacid (CaCO3). Immunogenicity and protective efficacy of the vaccine after buccal delivery was evaluated in the gnotobiotic pig model of human rotavirus (HRV) infection and diarrhea. Two doses of the vaccine were highly immunogenic and conferred strong protection against virus shedding and diarrhea upon challenge with a high dose of a virulent G1 HRV in gnotobiotic pigs. Those pigs vaccinated with the preserved film vaccine had significantly delayed onset of diarrhea; reduced duration and area under the curve of diarrhea; delayed onset of fecal virus shedding; and reduced duration and peak of fecal virus shedding titers compared to pigs in both the placebo and the reconstituted liquid oral RRV-TV vaccine groups. Associated with the strong protection, high titers of serum virus neutralization antibodies against each of the four RRV-TV mono-reassortants and G1 HRV-specific serum IgA and IgG antibodies, as well as intestinal IgA antibodies, were induced by the preserved film vaccine. These results demonstrated the effectiveness of our thermostable buccal film rotavirus vaccine and warrant further investigation into the promise of the novel technology in addressing drawbacks of the current live oral HRV vaccines.

Microbiota in viral infection and disease in humans and farm animals.

The influence of the microbiota on viral infection susceptibility and disease outcome is undisputable although varies among viruses. The purpose of understanding the interactions between microbiota, virus, and host is to identify practical, effective, and safe approaches that target microbiota for the prevention and treatment of viral diseases in humans and animals, as currently there are few effective and reliable antiviral therapies available. The initial step for achieving this goal is to gather clinical evidences, focusing on the viral pathogens-from human and animal studies-that have already been shown to interact with microbiota. The subsequent step is to identify mechanisms, through experimental evidences, to support the development of translational applications that target microbiota. In this chapter, we review evidences of virus infections altering microbiota and of microbiota enhancing or suppressing infectivity, altering host susceptibility to certain viral diseases, and influencing vaccine immunogenicity in humans and farm animals.

Defining the Specific Pathogen-Free State of Xenopus Using TaqMan Assays.

Colonies of valuable inbred and transgenic laboratory-reared Xenopus frogs maintained for research constitute naïve populations of animals susceptible to some opportunistic infectious diseases. Therefore, it is prudent to characterize any new animal acquisitions before introduction into an existing colony as a biosecurity measure to preclude the concurrent introduction of an infectious microorganism associated with the new animal(s). In addition, some pathogens of Xenopus, such as Chlamydia and Mycobacterium spp, are zoonotic diseases, placing frog aquarists at risk for acquiring an infection. Because it is not cost effective to test for all diseases of Xenopus frogs, we have defined a subset of prevalent infectious microorganisms and developed TaqMan polymerase chain reaction (PCR) assays to detect these agents. The specific pathogens in our test panel were selected from relatively recent publications where they reportedly caused morbidity and/or mortality in Xenopus laevis and/or X. tropicalis The assays herein do not constitute a comprehensive list of infectious diseases of Xenopus frogs. Therefore, a frog devoid of the infectious agents in our test panel are characterized as "specific pathogen-free." Three of the described quantitative polymerase chain reaction (qPCR) assays detect many species within their genus (i.e., qPCRs for ranaviruses, Chlamydia spp, and Cryptosporidia spp).