Biomarkers associated with vaccine-associated enhanced respiratory disease following influenza A virus infection in swine.

Influenza A virus (IAV) is a major pathogen in the swine industry. Whole-inactivated virus (WIV) vaccines in swine are highly effective against homologous viruses but provide limited protection to antigenically divergent viruses and may lead to vaccine-associated enhanced respiratory disease (VAERD) after heterologous infection. Although VAERD is reproducible in laboratory studies, clinical diagnosis is challenging, as it would require both knowledge of prior vaccine history and evidence of severe disease by assessment of pathologic lesions at necropsy following infection with a heterologous virus. The objective of this study was to identify potential biomarkers for VAERD for antemortem clinical diagnosis. Naïve pigs were split into two groups, and one group was vaccinated with IAV WIV vaccine. All pigs were then challenged with a heterologous virus to induce VAERD in the vaccinated group and necropsied at 5 days post infection (dpi). Blood was collected on 0, 1, 3, and 5 dpi, and assessed by hematology, plasma chemistry, acute phase proteins, and citrullinated H3 histone (CitH3) assays. Additionally, cytokine and CitH3 levels were assessed in bronchoalveolar lavage fluid (BALF) collected at necropsy. Compared to nonvaccinated challenged pigs, blood collected from vaccinated and challenged (V/C) pigs with VAERD had elevated white blood cells and neutrophils, elevated C-reactive protein and haptoglobin acute phase proteins, and elevated CitH3. In BALF, the proinflammatory cytokine IL-8 and CitH3 were elevated in V/C pigs. In conclusion, a profile of elevated white blood cells and neutrophils, elevated C-reactive protein and haptoglobin, and elevated CitH3 may be relevant for a clinical antemortem IAV VAERD diagnosis.

Bivalent hemagglutinin and neuraminidase influenza replicon particle vaccines protect pigs against influenza a virus without causing vaccine associated enhanced respiratory disease.

Alphavirus-derived RNA replicon particle (RP) vaccines represent the next generation of swine influenza A virus (IAV) vaccines, as they were shown to be safe, effective, and offer advantages over traditional vaccine platforms. IAV is a significant respiratory pathogen of swine and there is a critical need to improve current commercial swine IAV vaccine platforms. Adjuvanted whole inactivated virus (WIV) IAV swine vaccines provide limited heterologous protection and may lead to vaccine-associated enhanced respiratory disease (VAERD). This study investigated the ability of RP IAV hemagglutinin (HA) vaccines to avoid VAERD and evaluated experimental multivalent HA and neuraminidase (NA) RP vaccines. RP vaccines were formulated with HA or NA heterologous or homologous to the challenge virus in monovalent HA or HA and NA bivalent combinations (HA/NA bivalent). Pigs were vaccinated with an HA RP, HA/NA bivalent RP, or heterologous HA WIV, followed by IAV challenge and necropsy 5 days post infection. RP vaccines provided homologous protection from challenge and induced robust peripheral and local antibody responses. The RP vaccine did not induce VAERD after challenge with a virus containing the heterologous HA, in contrast to the traditional WIV vaccine. The HA monovalent and HA/NA bivalent RP vaccines showed superior protection compared to traditional WIV. Additionally, the RP platform allows greater flexibility to adjust HA and NA content to reflect circulating IAV in swine antigenic diversity.

Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in life.

The gastrointestinal microbiota begins to be acquired at birth and continually matures through early adolescence. Despite the relevance for gut health, few studies have evaluated the impact of pathobiont colonization of neonates on the severity of colitis later in life. LF82 is an adherent invasive E. coli strain associated with ileal Crohn's disease. The aim of this study was to evaluate the severity of dextran sodium sulfate (DSS)-induced colitis in mice following E. coli LF82 colonization. Gnotobiotic mice harboring the altered Schaedler flora (ASF) were used as the model. While E. coli LF82 is neither adherent nor invasive, it was been demonstrated that adult ASF mice colonized with E. coli LF82 develop more severe DSS-induced colitis compared to control ASF mice treated with DSS. Therefore, we hypothesized that E. coli LF82 colonization of neonatal ASF mice would reduce the severity of DSS-induced inflammation compared to adult ASF mice colonized with E. coli LF82. To test this hypothesis, adult ASF mice were colonized with E. coli LF82 and bred to produce offspring (LF82N) that were vertically colonized with LF82. LF82N and adult-colonized (LF82A) mice were given 2.0% DSS in drinking water for seven days to trigger colitis. More severe inflammatory lesions were observed in the LF82N + DSS mice when compared to LF82A + DSS mice, and were characterized as transmural in most of the LF82N + DSS mice. Colitis was accompanied by secretion of proinflammatory cytokines (IFNγ, IL-17) and specific mRNA transcripts within the colonic mucosa. Using 16S rRNA gene amplicon sequencing, LF82 colonization did not induce significant changes in the ASF community; however, minimal changes in spatial redistribution by fluorescent in situ hybridization were observed. These results suggest that the age at which mice were colonized with E. coli LF82 pathobiont differentially impacted severity of subsequent colitic events.

Vaccine-Associated Enhanced Respiratory Disease following Influenza Virus Infection in Ferrets Recapitulates the Model in Pigs.

Influenza A virus (IAV) causes respiratory disease in swine and humans. Vaccines are used to prevent influenza illness in both populations but must be frequently updated due to rapidly evolving strains. Mismatch between the circulating strains and the strains contained in vaccines may cause loss of efficacy. Whole inactivated virus (WIV) vaccines with adjuvant, utilized by the swine industry, are effective against antigenically similar viruses; however, vaccine-associated enhanced respiratory disease (VAERD) may happen when the WIV is antigenically mismatched with the infecting virus. VAERD is a repeatable model in pigs, but had yet to be experimentally demonstrated in other mammalian species. We recapitulated VAERD in ferrets, a standard benchmark animal model for studying human influenza infection, in a direct comparison to VAERD in pigs. Both species were vaccinated with WIV with oil-in-water adjuvant containing a δ-1 H1N2 (1B.2.2) derived from the pre-2009 human seasonal lineage, then challenged with a 2009 pandemic H1N1 (H1N1pdm09, 1A.3.3.2) 5 weeks after vaccination. Nonvaccinated and challenged groups showed typical signs of influenza disease, but the mismatched vaccinated and challenged pigs and ferrets showed elevated clinical signs, despite similar viral loads. VAERD-affected pigs exhibited a 2-fold increase in lung lesions, while VAERD-affected ferrets showed a 4-fold increase. Similar to pigs, antibodies from VAERD-affected ferrets preferentially bound to the HA2 domain of the H1N1pdm09 challenge strain. These results indicate that VAERD is not limited to pigs, as demonstrated here in ferrets, and the need to consider VAERD when evaluating new vaccine platforms and strategies. IMPORTANCE We demonstrated the susceptibility of ferrets, a laboratory model species for human influenza A virus research, to vaccine-associated enhanced respiratory disease (VAERD) using an experimental model previously demonstrated in pigs. Ferrets developed clinical characteristics of VAERD very similar to that in pigs. The hemagglutinin (HA) stalk is a potential vaccine target to develop more efficacious, broadly reactive influenza vaccine platforms and strategies. However, non-neutralizing antibodies directed toward a conserved epitope on the HA stalk induced by an oil-in-water, adjuvanted, whole influenza virus vaccine were previously shown in VAERD-affected pigs and were also identified here in VAERD-affected ferrets. The induction of VAERD in ferrets highlights the potential risk of mismatched influenza vaccines for humans and the need to consider VAERD when designing and evaluating vaccine strategies.

Campylobacter jejuni persistently colonizes gnotobiotic altered Schaedler flora C3H/HeN mice and induces mild colitis.

Campylobacter jejuni is a major cause of food-borne human bacterial gastroenteritis but animal models for C. jejuni mediated disease remain limited because C. jejuni poorly colonizes immunocompetent, conventionally-reared (Conv-R) mice. Thus, a reliable rodent model (i.e. persistent colonization) is desirable in order to evaluate C. jejuni-mediated gastrointestinal disease and mechanisms of pathogenicity. As the nature and complexity of the microbiota likely impacts colonization resistance for C. jejuni, Conv-R and gnotobiotic C3H/HeN mice were used to evaluate the persistence of C. jejuni colonization and development of disease. A total of four C. jejuni isolates readily and persistently colonized ASF mice and induced mild mucosal inflammation in the proximal colon, but C. jejuni did not stably colonize nor induce lesions in Conv-R mice. This suggests that the pathogenesis of C. jejuni is influenced by the microbiota, and that ASF mice offer a reproducible model to study the influence of the microbiota on the ability of C. jejuni to colonize the gut and to mediate gastroenteritis.

Pathogenic and non-pathogenic Escherichia coli colonization and host inflammatory response in a defined microbiota mouse model.

Most Escherichia coli strains in the human intestine are harmless. However, enterohemorrhagic Ecoli (EHEC) is a foodborne pathogen that causes intestinal disease in humans. Conventionally reared (CONV) mice are inconsistent models for human infections with EHEC because they are often resistant to Ecoli colonization, in part due to their gastrointestinal (GI) microbiota. Although antibiotic manipulation of the mouse microbiota has been a common means to overcome colonization resistance, these models have limitations. Currently, there are no licensed treatments for clinical EHEC infections and, thus, new tools to study EHEC colonization need to be developed. Here, we used a defined microbiota mouse model, consisting of the altered Schaedler flora (ASF), to characterize intestinal colonization and compare host responses following colonization with EHEC strain 278F2 or non-pathogenic Ecoli strain MG1655. Significantly higher (P<0.05) levels of both strains were found in feces and cecal and colonic contents of C3H/HeN ASF compared to C3H/HeN CONV mice. GI inflammation was significantly elevated (P<0.05) in the cecum of EHEC 278F2-colonized compared to E. coli MG1655-colonized C3H/HeN ASF mice. In addition, EHEC 278F2 differentially modulated inflammatory-associated genes in colonic tissue of C3H/HeN ASF mice compared to E. coli MG1655-colonized mice. This approach allowed for prolonged colonization of the murine GI tract by pathogenic and non-pathogenic Ecoli strains, and for evaluation of host inflammatory processes. Overall, this system can be used as a powerful tool for future studies to assess therapeutics, microbe-microbe interactions, and strategies for preventing EHEC infections.

Orally administered extract from Prunella vulgaris attenuates spontaneous colitis in mdr1a(-/-) mice.

AIM: To investigate the ability of a Prunella vulgaris (P. vulgaris) ethanolic extract to attenuate spontaneous typhlocolitis in mdr1a(-/-) mice. METHODS: Vehicle (5% ethanol) or P. vulgaris ethanolic extract (2.4 mg/d) were administered daily by oral gavage to mdr1a(-/-) or wild type FVB(WT) mice from 6 wk of age up to 20 wk of age. Clinical signs of disease were noted by monitoring weight loss. Mice experiencing weight loss in excess of 15% were removed from the study. At the time mice were removed from the study, blood and colon tissue were collected for analyses that included histological evaluation of lesions, inflammatory cytokine levels, and myeloperoxidase activity. RESULTS: Administration of P. vulgaris extracts to mdr1a(-/-) mice delayed onset of colitis and reduced severity of mucosal inflammation when compared to vehicle-treated mdr1a(-/-) mice. Oral administration of the P. vulgaris extract resulted in reduced (P < 0.05) serum levels of IL-10 (4.6 ± 2 vs 19.4 ± 4), CXCL9 (1319.0 ± 277 vs 3901.0 ± 858), and TNFα (9.9 ± 3 vs 14.8 ± 1) as well as reduced gene expression by more than two-fold for Ccl2, Ccl20, Cxcl1, Cxcl9, IL-1α, Mmp10, VCAM-1, ICAM, IL-2, and TNFα in the colonic mucosa of mdr1a(-/-) mice compared to vehicle-treated mdr1a(-/-) mice. Histologically, several microscopic parameters were reduced (P < 0.05) in P. vulgaris-treated mdr1a(-/-) mice, as was myeloperoxidase activity in the colon (2.49 ± 0.16 vs 3.36 ± 0.06, P < 0.05). The numbers of CD4(+) T cells (2031.9 ± 412.1 vs 5054.5 ± 809.5) and germinal center B cells (2749.6 ± 473.7 vs 4934.0 ± 645.9) observed in the cecal tonsils of P. vulgaris-treated mdr1a(-/-) were significantly reduced (P < 0.05) from vehicle-treated mdr1a(-/-) mice. Vehicle-treated mdr1a(-/-) mice were found to produce serum antibodies to antigens derived from members of the intestinal microbiota, indicative of severe colitis and a loss of adaptive tolerance to the members of the microbiota. These serum antibodies were greatly reduced or absent in P. vulgaris-treated mdr1a(-/-) mice. CONCLUSION: The anti-inflammatory activity of P. vulgaris ethanolic extract effectively attenuated the severity of intestinal inflammation in mdr1a(-/-) mice.

The Altered Schaedler Flora: Continued Applications of a Defined Murine Microbial Community.

The gastrointestinal (GI) microbiota forms a mutualistic relationship with the host through complex and dynamic interactions. Because of the complexity and interindividual variation of the GI microbiota, investigating how members of the microbiota interact with each other, as well as with the host, is daunting. The altered Schaedler flora (ASF) is a model community of eight microorganisms that was developed by R.P. Orcutt and has been in use since the late 1970s. The eight microorganisms composing the ASF were all derived from mice, can be cultured in vitro, and are stably passed through multiple generations (at least 15 years or more by the authors) in gnotobiotic mice continually bred in isolator facilities. With the limitations associated with conventional, mono- or biassociated, and germfree mice, use of mice colonized with a consortium of known bacteria that naturally inhabit the murine gut offers a powerful system to investigate mechanisms governing host-microbiota relationships, and how members of the GI microbiota interact with one another. The ASF community offers significant advantages to study homeostatic as well as disease-related interactions by taking advantage of a well-defined, limited community of microorganisms. For example, quantification and spatial distribution of individual members, microbial genetic manipulation, genomic-scale analysis, and identification of microorganism-specific host immune responses are all achievable using the ASF model. This review compiles highlights associated with the 37-year history of the ASF, including descriptions of its continued use in biomedical research to elucidate the complexities of host-microbiome interactions in health and disease.