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.

Correlation of Feline Coronavirus Shedding in Feces with Coronavirus Antibody Titer

Background: Feline coronavirus (FCoV) infection is ubiquitous in multi-cat households. Responsible for the continuous presence are cats that are chronically shedding a high load of FCoV. The aim of the study was to determine a possible correlation between FCoV antibody titer and frequency and load of fecal FCoV shedding in cats from catteries. Methods: Four fecal samples from each of 82 cats originating from 19 German catteries were examined for FCoV viral loads by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Additionally, antibody titers were determined by an immunofluorescence assay. Results: Cats with antibodies were more likely to be FCoV shedders than non-shedders, and there was a weak positive correlation between antibody titer and mean fecal virus load (Spearman r = 0.2984; p = 0.0072). Antibody titers were significantly higher if cats shed FCoV more frequently throughout the study period (p = 0.0063). When analyzing only FCoV shedders, cats that were RT-qPCR-positive in all four samples had significantly higher antibody titers (p = 0.0014) and significantly higher mean fecal virus loads (p = 0.0475) than cats that were RT-qPCR-positive in only one, two, or three samples. Conclusions: The cats’ antibody titers correlate with the likelihood and frequency of FCoV shedding and fecal virus load. Chronic shedders have higher antibody titers and shed more virus. This knowledge is important for the management of FCoV infections in multi-cat environments, but the results indicate that antibody measurement cannot replace fecal RT-qPCR.

Mucosal Immune Response to Feline Enteric Coronavirus Infection

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into naïve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFNγ responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.

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.

Rectal Immunization with Rotavirus Virus-Like Particles Induces Systemic and Mucosal Humoral Immune Responses and Protects Mice against Rotavirus Infection

ABSTRACT To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.

Capped RNA Transcripts of Full-Length cDNA Clones of Swine Hepatitis E Virus Are Replication Competent When Transfected into Huh7 Cells and Infectious When Intrahepatically Inoculated into Pigs

ABSTRACT Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.

Pathogenesis of Canine Parvovirus Enteritis: Sequential Virus Distribution and Passive Immunization Studies

After oral inoculation, the sequential distribution of canine parvovirus was studied in 14 nine-week-old seronegative beagle dogs. Two or three dogs were necropsied on days 1 through 6 after inoculation. Tissues were collected for virus isolation, immunofluorescence testing, and light microscopy. Virus was isolated from, and fluorescent cells were seen in the tonsil, retropharyngeal and mesenteric lymph nodes one and two days after inoculation. Virus infection of systemic and intestinal lymphoid tissues occurred as early as three days after inoculation and was associated with viremia. Intestinal epithelial infection was first seen four days after oral inoculation. All dogs were viremic before intestinal epithelial infection was found. Fecal virus excretion first occurred four days after oral virus inoculation. Intestinal virus infection and lesions became progressively more severe between four and six days after inoculation. The severity of intestinal lesions was variable and related to the severity of systemic lymphoid tissue lesions and the magnitude and duration of viremia. Four littermates of virus-infected dogs were passively immunized against canine parvovirus with convalescent canine serum 24 hours after oral virus inoculation. Neither clinical signs, lymphopenia, nor fecal virus excretion occurred in passively immunized dogs. Intestinal epithelial infection was not demonstrable by immunofluorescence testing when passively immunized dogs were necropsied four, five, and six days after virus inoculation.