Keratinocyte-specific deletion of SHARPIN induces atopic dermatitis-like inflammation in mice.

Spontaneous mutations in the SHANK-associated RH domain interacting protein (Sharpin) resulted in a severe autoinflammatory type of chronic proliferative dermatitis, inflammation in other organs, and lymphoid organ defects. To determine whether cell-type restricted loss of Sharpin causes similar lesions, a conditional null mutant was created. Ubiquitously expressing cre-recombinase recapitulated the phenotype seen in spontaneous mutant mice. Limiting expression to keratinocytes (using a Krt14-cre) induced a chronic eosinophilic dermatitis, but no inflammation in other organs or lymphoid organ defects. The dermatitis was associated with a markedly increased concentration of serum IgE and IL18. Crosses with S100a4-cre resulted in milder skin lesions and moderate to severe arthritis. This conditional null mutant will enable more detailed studies on the role of SHARPIN in regulating NFkB and inflammation, while the Krt14-Sharpin-/- provides a new model to study atopic dermatitis.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes.

Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease.

Formulation of a killed whole cell pneumococcus vaccine - effect of aluminum adjuvants on the antibody and IL-17 response.

BACKGROUND: Streptococcus pneumoniae causes widespread morbidity and mortality. Current vaccines contain free polysaccharides or protein-polysaccharide conjugates, and do not induce protection against serotypes that are not included in the vaccines. An affordable and broadly protective vaccine is very desirable. The goal of this study was to determine the optimal formulation of a killed whole cell pneumococcal vaccine with aluminum-containing adjuvants for intramuscular injection. METHODS: Four aluminium-containing adjuvants were prepared with different levels of surface phosphate groups resulting in different adsorptive capacities and affinities for the vaccine antigens. Mice were immunized three times and the antigen-specific antibody titers and IL-17 responses in blood were analyzed. RESULTS: Although all adjuvants induced significantly higher antibody titers than antigen without adjuvant, the vaccine containing aluminum phosphate adjuvant (AP) produced the highest antibody response when low doses of antigen were used. Aluminum hydroxide adjuvant (AH) induced an equal or better antibody response at high doses compared with AP. Vaccines formulated with AH, but not with AP, induced an IL-17 response. The vaccine formulated with AH was stable and retained full immunogenicity when stored at 4°C for 4 months. CONCLUSIONS: Antibodies are important for protection against systemic streptococcal disease and IL-17 is critical in the prevention of nasopharyngeal colonization by S. pneumoniae in the mouse model. The formulation of the whole killed bacterial cells with AH resulted in a stable vaccine that induced both antibodies and an IL-17 response. These experiments underscore the importance of formulation studies with aluminium containing adjuvants for the development of stable and effective vaccines.

Expression of chitinase-like proteins in the skin of chronic proliferative dermatitis (cpdm/cpdm) mice.

Mammalian chitinase-like proteins belong to a family of proteins structurally related to chitinases but devoid of enzymatic activity. They have a postulated role in remodeling of extracellular matrix and defense mechanisms against chitin-containing pathogens. The expression of these proteins is increased in parasitic infections and allergic airway disease, but their expression in dermatitis has not been examined. The mRNA expression of two chitinase 3-like (Chi3L) proteins, Chi3L3 (Ym1) and Chi3L4 (Ym2), was determined in the skin of normal mice, chronic proliferative dermatitis (cpdm/cpdm) mutant mice and mice with experimentally induced contact hypersensitivity reaction. The localization of Chi3L3 and Chi3L4 proteins in cells was determined by fluorescence microscopy of double-labeled frozen sections of skin, and confirmed in vitro by stimulation of macrophages and mast cells with cytokines. Quantitative RT-PCR demonstrated a 976-fold increase of Chi3l4 mRNA expression and a 24-fold increase of Chi3l3 mRNA expression in the skin of cpdm/cpdm mice. Their expression was also increased in the ears of mice with 2,4-dinitrofluorobenzene-induced contact hypersensitivity, but the increase was greater for Chi3l3 mRNA (51-fold) than Chi3l4 mRNA (32-fold). Western blot analysis with an antibody against Chi3L3 and Chi3L4 confirmed the increased amount of these proteins in the skin of cpdm/cpdm mice. Two-color immunofluorescence identified macrophages, dendritic cells and mast cells as cellular sources of Chi3L3 and Chi3L4 proteins. Eosinophils and neutrophils did not contain detectable concentrations of these proteins. Treatment of macrophages and mast cells in vitro with interleukin-4 induced expression of Chi3l3 and Chi3l4 mRNA.

Effect of age on immune parameters and the immune response of dogs to vaccines: a cross-sectional study.

The evaluation of anti-aging intervention strategies in dogs would benefit from reliable quantitative biomarkers of aging. In the present study, the expression of various immune parameters was measured in young and old dogs to identify potential biomarkers of aging. The second goal of the study was to determine the effect of age on the immune response to vaccines. The immune function, including the antibody response to vaccines, was determined in 32 young adult (3.15+/-0.8 years of age) and 33 old dogs (12.1+/-1.3 years of age) of various breeds. Old dogs had a significantly lower lymphocyte proliferative response and a lower percentage of CD4+ T cells and CD45R+/CD4+ T cells, and a higher percentage of CD8+ T cells and a higher concentration of serum and salivary IgA. The most significant differences (P<0.001) occurred in the lymphocyte proliferative responses to ConA and PHA, the CD4:CD8 ratio, and the percentage of CD45R+/CD4+ T cells suggesting that these parameters are potential biomarkers of aging. There was no difference in the percentage of total T and B lymphocytes and the concentration of serum IgM and IgG. Both groups of dogs had protective titers against distemper virus, parvovirus and rabies virus before annual revaccination. The pre-vaccination titer against rabies virus was higher in the old dogs than in the young dogs, and there were no differences in post-vaccination titers against any of the viruses. This suggests that annual vaccination protocols provide adequate protection for old dogs.

Effect of vaccination on serum concentrations of total and antigen-specific immunoglobulin E in dogs.

OBJECTIVE: To determine the effect of vaccination on serum concentrations of total and antigen-specific IgE in dogs. ANIMALS: 20 female Beagles. PROCEDURE: Groups of 5 dogs each were vaccinated repeatedly between 8 weeks and 4 years of age with a multivalent and rabies vaccine, a multivalent vaccine only, or a rabies vaccine only. A fourth group of 5 dogs served as unvaccinated controls. Serum concentrations of total immunoglobulins and antigen-specific IgE were determined following vaccination. RESULTS: -The multivalent vaccine had little effect on serum total IgE concentrations. The concentration of IgE increased slightly following vaccination for rabies at 16 weeks and 1 year of age and increased greatly after vaccination at 2 and 3 years of age in most dogs, with a distinct variation between individual dogs. Vaccination had no effect on serum concentrations of IgA, IgG, and IgM as measured at 2 and 3 years of age. The rabies vaccine contained aluminum adjuvant in contrast to the multivalent vaccine. An increase of IgE that was reactive with vaccine antigens, including bovine serum albumin and bovine fibronectin, was detected in some of the dogs vaccinated for rabies. There was no significant correlation between serum concentrations of total IgE and antigen-specific IgE following vaccination. Serum total IgE concentration rapidly returned to preimmunization concentrations in most dogs, but high concentrations of antigen-specific IgE persisted. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination of dogs for rabies increases serum concentrations of total IgE and induces IgE specific for vaccine antigens, including tissue culture residues. Vaccination history should be considered in the interpretation of serum total IgE concentrations.