An RSV Live-Attenuated Vaccine Candidate Lacking G Protein Mucin Domains Is Attenuated, Immunogenic, and Effective in Preventing RSV in BALB/c Mice.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. METHODS: Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. RESULTS: Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. CONCLUSIONS: Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist, SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection.

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.

Comparison of a commercially available oral nutritional supplement and intravenous fluid therapy for dehydration in dairy calves.

Calf scours is a primary cause of morbidity and mortality in the dairy industry. Effective treatments are needed to minimize death, maximize welfare, and maintain growth and productivity. The objective of this trial was to compare the efficacy of a commercially available nutritional supplement (Diaque, Boehringer-Ingelheim Vetmedica Inc., St. Joseph, MO) and i.v. lactated Ringer's solution (LRS) in rehydrating, preventing acidemia, and correcting electrolyte imbalances in an experimental model for calf scours. Twenty-four colostrum-fed suckling dairy calves were used in a modified crossover design. An osmotic diarrhea was induced by orally feeding commercial milk replacer modified with high level of sucrose to create a hypertonic milk solution, and administering oral hydrochlorothiazide and spironolactone for 48 h. The intention was to create a challenge sufficient to result in moderately dehydrated, standing calves without producing severe depression or loss of suckle. The efficacy of i.v. fluid therapy and a commercial nutritional supplement were subsequently compared for reversing the effects of the diarrheal disease. Treatment A consisted of administering the nutritional supplement according to label directions (100 g in 1.9 L of warm water, 3 times a day), and treatment B consisted of i.v. LRS (2 L, once a day). Clinical signs and laboratory results were obtained once daily by a blinded observer. The induction method was effective in creating the desired effect, as demonstrated by weight loss and subjective health and hydration scores. Both treatment groups experienced increases in body weight, base excess, and bicarbonate, and decreases in total protein and packed cell volume following treatment. Both i.v. LRS and Diaque are effective methods to correct hypovolemia and control derangements in acid-base status in calves with diarrhea and dehydration.

Reproductive Disorders in Horses.

Reproductive disease is relatively common in the horse, resulting in a variable, yet significant, economic impact on individual horsemen as well as the entire industry. Diverse expertise from the veterinary community ensures and improves individual and population health of the horse. From a pathology and diagnostics perspective, this review provides a comprehensive overview of pathology of the male and female equine reproductive tract. Recognition by clinical and gross features is emphasized, although some essential histologic parameters are included, as appropriate. Where relevant, discussion of ancillary diagnostic tests and approaches are included for some diseases and lesions.

Antibody levels following vaccination of beef calves with a tetanus toxoid.

Tetanus is a preventable, yet often fatal, disease affecting many species, including beef cattle. Vaccination for tetanus is recommended for calves at high risk of disease, but typical beef cattle management practices often make adherence to vaccine manufacturers' guidance for a second (booster) dose of vaccine difficult. This study examined the antibody response following a single dose of tetanus toxoid, as well as following booster vaccination at various intervals. Anti-tetanus IgG antibodies were detectable 25 days (D25) after a single dose, and rose following booster at either D25 D109 after initial vaccination. Antibody levels then declined numerically from D109 to D179 for calves boostered at D25 but rose on D179 for those receiving a second dose on D109. The relatively rapid response in IgG production, even in the absence of a booster vaccine, may suggest value in vaccinating calves for tetanus at time of greatest risk, even if a booster cannot be administered. The study also provides support for priming of the immune response lasting at least until D109 after primary immunization.

Intranasal Vaccination with a Respiratory-Syncytial-Virus-Based Virus-like Particle Displaying the G Protein Conserved Region Induces Severe Weight Loss and Pathology upon Challenge with Wildtype Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population.

Enhancing Anti-G Antibody Induction by a Live Single-Cycle Prefusion F-Expressing RSV Vaccine Improves In Vitro and In Vivo Efficacy.

The human respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease, and a vaccine is not available. We previously reported a novel live vaccine expressing prefusion-stabilized fusion protein (preF) in place of the native F protein (RSV-preFΔCT). As preF is non-functional, RSV-preFΔCT was amplified in a production line expressing a functional substitute, and exhibited a single-cycle replication phenotype, which holds several unique potential advantages. RSV-preFΔCT prevented shedding and lung pathology after viral challenge in mice, but induced low levels of anti-attachment protein (G) antibodies (Abs). Given the significant contributions of anti-G Abs toward disease prevention, we generated modifications to RSV-preFΔCT in an effort to induce higher anti-G Ab levels. The Ab levels were monitored after the prime-boost vaccination of mice with modified vaccines. The most successful modification for enhancing induced anti-G Abs was seen with the placement of G in the first genome position. This vaccine also reduced the pathology after challenge with a high dose of wt RSV, and outperformed the sera from wt RSV-vaccinated mice in in vitro neutralization. Thus, raising the anti-G Ab levels induced by RSV-preFΔCT enhanced efficacy in vitro and in vivo, and constitutes an important next step in developing a live, single-cycle, efficacious vaccine for the human population.

Corrigendum: Genetic inactivation of Chlamydia trachomatis inclusion membrane protein CT228 alters MYPT1 recruitment, extrusion production, and longevity of infection.

[This corrects the article DOI: 10.3389/fcimb.2018.00415.].

A live single-cycle RSV vaccine expressing prefusion F protein.

Live-attenuated Respiratory syncytial virus (RSV) vaccines given intranasally have potential to provide comprehensive protection, including lung-resident immunity. It has however proven challenging to impart both sufficient safety and efficacy in a vaccine. To achieve the latter, we used a trans-complementing approach to generate live single-cycle RSV vaccines expressing the prefusion form (preF) of the viral fusion protein (F), either membrane-anchored or secreted. Both viruses were tested for their ability to induce a protective immune response in mice after intranasal prime-boost vaccination. The secreted preF vaccine failed to induce a protective response. The anchored preF vaccine induced anti-preF antibodies and antiviral T cells, and protected mice from lung pathology and viral shedding after challenge. Neither vaccine induced anti-G antibodies, for reasons unknown. In spite of the latter and single-cycle replication, the membrane-anchored preF vaccine was protective and demonstrates potential for development of an efficacious live vaccine with a stable safety phenotype.

Severe anemia associated with Mycoplasma wenyonii infection in a mature cow.

The clinical findings, diagnostic tests, and treatment of clinical anemia in a mature Angus cow infected with the hemoplasma Mycoplasma wenyonii are described. Mycoplasma wenyonii has been previously reported to cause clinical anemia in young or splenectomized cattle; however, infection has not been associated with severe anemia in mature animals.

The geropathology of organ-specific aging.

Aging is a complex multidimensional process of progressive decline affecting multiple organ systems by a number of processes that are still not well understood. While many studies have focused on the approach of studying aging across multiple organs, assessment of the contribution of individual organs to overall aging processes is under appreciated. The ability to study and compare organs in the context of organismal aging has been documented recently using a geropathology grading platform in laboratory mice. This concept consists of identifying and grading age-related histologic lesions within organs to generate a quantitative lesion score for each organ, which is representative of the presence and degree of organ-related pathology, and can be compared to scores from other organs examined. This geropathology approach provides a powerful tool to elucidate the basic mechanisms of aging in multiple organs, as well as the response of organs to therapeutic interventions. Furthermore, ongoing work with the concept has expanded and adapted the geropathology grading system to other preclinical animal model species that are commonly used to understand disease associated phenotypes in aging humans, ultimately adding to the utility of the concept.

Oklahoma Nathan Shock Aging Center - assessing the basic biology of aging from genetics to protein and function.

The Oklahoma Shock Nathan Shock Center is designed to deliver unique, innovative services that are not currently available at most institutions. The focus of the Center is on geroscience and the development of careers of young investigators. Pilot grants are provided through the Research Development Core to junior investigators studying aging/geroscience throughout the USA. However, the services of our Center are available to the entire research community studying aging and geroscience. The Oklahoma Nathan Shock Center provides researchers with unique services through four research cores. The Multiplexing Protein Analysis Core uses the latest mass spectrometry technology to simultaneously measure the levels, synthesis, and turnover of hundreds of proteins associated with pathways of importance to aging, e.g., metabolism, antioxidant defense system, proteostasis, and mitochondria function. The Genomic Sciences Core uses novel next-generation sequencing that allows investigators to study the effect of age, or anti-aging manipulations, on DNA methylation, mitochondrial genome heteroplasmy, and the transcriptome of single cells. The Geroscience Redox Biology Core provides investigators with a comprehensive state-of-the-art assessment of the oxidative stress status of a cell, e.g., measures of oxidative damage and redox couples, which are important in aging as well as many major age-related diseases as well as assays of mitochondrial function. The GeroInformatics Core provides investigators assistance with data analysis, which includes both statistical support as well as analysis of large datasets. The Core also has developed number of unique software packages to help with interpretation of results and discovery of new leads relevant to aging. In addition, the Geropathology Research Resource in the Program Enhancement Core provides investigators with pathological assessments of mice using the recently developed Geropathology Grading Platform.

Confirmation of Amblyomma americanum (Acari: Ixodidae) as a vector for Cytauxzoon felis (Piroplasmorida: Theileriidae) to domestic cats.

Amblyomma americanum was confirmed as a competent vector in the transmission of Cytauxzoon felis to domestic cats. Infection with C. felis was produced and replicated in four domestic felines by the bite of A. americanum adults that were acquisition fed as nymphs on a domestic cat that survived cytauxzoonosis. Numerous attempts to transmit C. felis with Dermacentor variabilis at the same time were not successful. All cats upon which infected A. americanum were transmission fed exhibited disease typical of cytauxzoonosis, and the eitiologic agent's presence was confirmed. Clinical signs including fever, inappetence, depression, and lethargy were observed beginning 13 d postinfestation. Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. Rectal temperatures of the C. felis-infected principal cats fluctuated from high to subnormal before returning to the normal range. Clinical signs of cytauxzoonsis improved by 24 d postinfestation in all but one cat, with survivors remaining parasitemic and subclinically infected with C. felis. Unengorged A. americanum and D. variabilis were collected from wild habitats to determine the minimum infection rate of C. felis in ticks from an enzootic area. Infection of C. felis was found only in wild-collected A. americanum. The minimum infection rate of C. felis in A. americanum was 0.5% (one of 178) in males, 0.8% (three of 393) in nymphs, and 1.5% (three of 197) in females. We found no wild-collected D. variabilis infected with C. felis. Our results confirm that A. americanum is a primary vector of C. felis.

Production of Neutrophil Extracellular Traps Contributes to the Pathogenesis of Francisella tularemia.

Francisella tularensis(Ft) is a highly virulent, intracellular Gram-negative bacterial pathogen. Acute Ft infection by aerosol route causes pneumonic tularemia, characterized by nodular hemorrhagic lesions, neutrophil-predominant influx, necrotic debris, fibrin deposition, and severe alveolitis. Ft suppresses activity of neutrophils by impairing their respiratory burst and phagocytic activity. However, the fate of the massive numbers of neutrophils recruited to the infection site is unclear. Here, we show that Ft infection resulted in prominent induction of neutrophil extracellular traps (NETs) within damaged lungs of mice infected with the live attenuated vaccine strain of Ft(Ft-LVS), as well as in the lungs of domestic cats and rabbits naturally infected with Ft. Further, Ft-LVS infection increased lung myeloperoxidase (MPO) activity, which mediates histone protein degradation during NETosis and anchors chromatin scaffolds in NETs. In addition, Ft infection also induced expression of peptidylarginine deiminase 4, an enzyme that causes citrullination of histones during formation of NETs. The released NETs were found largely attached to the alveolar epithelium, and disrupted the thin alveolar epithelial barrier. Furthermore, Ft infection induced a concentration-dependent release of NETs from neutrophils in vitro. Pharmacological blocking of MPO reduced Ft-induced NETs release, whereas addition of H2O2 (a substrate of MPO) significantly augmented NETs release, thus indicating a critical role of MPO in Ft-induced NETs. Although immunofluorescence and electron microscopy revealed that NETs could efficiently trap Ft bacteria, NETs failed to exert bactericidal effects. Taken together, these findings suggest that NETs exacerbate tissue damage in pulmonary Ft infection, and that targeting NETosis may offer novel therapeutic interventions in alleviating Ft-induced tissue damage.

E. coli O157:H7 catabolism of intestinal mucin-derived carbohydrates and colonization.

E. coli O157:H7 colonizes the bovine intestine, can contaminate food through fecal shedding, and causes human diarrheal and systemic illnesses. Catabolism of particular carbohydrates by E. coli has been found to be important for intestinal colonization of mice. In this study, we assessed whether catabolism of two mucin-derived carbohydrates are important for E. coli O157:H7 colonization of adult cattle. This was accomplished by competitively co-colonizing streptomycin-treated adult cattle with a wild-type strain of E. coli O157:H7 and isogenic mutants in catabolic pathways for mucin-derived carbohydrates N-acetylgalactosamine or l-fucose. Both mutants colonized poorly compared to the wild-type during the initiation stage of colonization (days 0-6). During the maintenance stage of colonization (days 7-15), the mutant unable to use N-acetylgalactosamine did not show a colonization defect, whereas the strain unable to use fucose had a significant colonization defect. These results support the concept that growth and colonization of E. coli O157:H7 in the bovine rectum has a nutritional basis, with a nutrient preference for l-fucose over N-acetylgalactosamine.

Hypertrophic cardiomyopathy in two captive Bennett's wallabies (Macropus rufogriseus rufogriseus).

Hypertrophic cardiomyopathy (HCM) was diagnosed during postmortem examination of 2 captive adult Bennett's wallabies (Macropus rufogriseus rufogriseus). The wallabies were members of a mob (herd) of 3 wallabies, and 2 died spontaneously without clinical signs of heart failure being detected. Gross lesions in both cases included marked concentric hypertrophy of the left ventricle, pulmonary edema, and multifocal hemorrhage and subcutaneous edema of the hind limbs. Histologic lesions of the heart were limited to mild cardiac myofiber disarray and marked cardiac myofiber hypertrophy. A specific etiology for the HCM was not determined in either animal. The cardiac changes are similar to the left ventricular hypertrophy previously described in kangaroos.

Transmission of Cytauxzoon felis to a domestic cat by Amblyomma americanum.

Cytauxzoon felis was transmitted to a domestic cat by Amblyomma americanum. The infection was produced by the bite of A. americanum adults that were acquisition fed as nymphs on a domestic cat that naturally survived infection of C. felis. Fever, inappetence, depression, and lethargy were first noted 11 days post-infestation (dpi). Pale mucus membranes, splenomegaly, icterus, and dyspnea were also observed during the course of the disease. The body temperature of the experimentally infected C. felis cat was subnormal from 16 dpi until 24 dpi when it returned to within normal limits. All clinical signs of cytauxzoonsis began to resolve by 23 dpi when the cat became subclinically infected with C. felis. The cat developed a marked, regenerative anemia beginning by 13 dpi and reached a nadir at 20 dpi before recovering. A moderate neutrophilia and marked lymphocytosis also developed between 18 and 26 dpi. Schizonts of C. felis were observed in spleen aspirates of the infected cat at 15 dpi. DNA of C. felis was amplified by real-time PCR starting 17 dpi and piroplasms of C. felis were first noted by light microscopy 18 dpi. Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus were also tested in a similar manner at the same time but did not transmit C. felis. Prior to the present study, only D. variabilis had been shown experimentally to transmit infection of C. felis. This is the first report of C. felis being transmitted by A. americanum. The transmission of C. felis infection from one domestic cat to another indicates that domestic cats subclinically infected with C. felis may be a reservoir of infection for naive domestic cats.

Mannheimia haemolytica IgA-specific proteases.

Mannheimia haemolytica colonizes the nasopharynx of cattle and can cause severe fibrinous pleuropneumonia. IgA proteases are metalloendopeptidases released by bacteria that cleave IgA, enhancing colonization of mucosa. The objectives of these studies were to characterize M. haemolytica IgA1 and IgA2 proteases in vitro and in silico, to clone and sequence the genes for these proteases, and to demonstrate immunogenicity of components of the entire IgA protease molecule. Both IgA protease genes were cloned, expressed, and sequenced. Sequences were compared to other published sequences. Components were used to immunize mice to determine immunogenicity. Sera from healthy cattle and cattle that recovered from respiratory disease were examined for antibodies to IgA proteases. In order to assay the cleavage of bovine IgA with IgA1 protease, M. haemolytica culture supernatant was incubated with bovine IgA. Culture supernatant cleaved purified bovine IgA in the presence of ZnCl2. Both IgA proteases contain three domains, 1) IgA peptidase, 2) PL1_Passenger_AT and 3) autotransporter. IgA1 and IgA2 peptidases have molecular weights of 96.5 and 87 kDa, respectively. Convalescent bovine sera with naturally high anti-M. haemolytica antibody titers had high antibodies against all IgA1 & IgA2 protease components. Mouse immunizations indicated high antibodies to the IgA peptidases and autotransporters but not to PL1_Passenger_AT. These data indicate that M. haemolytica produces two IgA proteases that are immunogenic, can cleave bovine IgA, and are produced in vivo, as evidenced by antibodies in convalescent bovine sera. Further studies could focus on IgA protease importance in pathogenesis and immunity.