Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb), designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs) from these species relative to cells incubated with an isotype control (p<0.001). In addition, the mAb induced significant nitric oxide (p<0.0001) release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001) IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Preservation of phenotype in an organotypic cell culture model of a recessive keratinization defect of Norfolk terrier dogs.

The purpose of this study is to reproduce in vitro a recessive keratinization defect of Norfolk terrier dogs characterized by a lack of keratin 10 (K10) production. Keratinocytes from skin biopsy samples of four normal dogs and two affected dogs were cultured organotypically with growth factor-supplemented media in order to stimulate cornification. The cultured epidermis from the normal dogs closely resembled the normal epidermis in vivo and cornified. The cultured epidermis from the affected dogs displayed many phenotypic alterations identified in skin biopsies from dogs with this heritable defect. Immunohistochemistry and immunoblotting showed a marked decrease in K10 from the cultures of the affected keratinocytes, compared to that in K10 from the cultures of the normal keratinocytes. Real-time reverse transcription polymerase chain reaction quantitation showed a 31-fold decrease in K10, a 1.75-fold increase in K1 and a 136-fold increase in K2e between the affected and the normal epidermis. Organotypic keratinocytes showed a 241-fold decrease in K10, a 31-fold decrease in K1 and a 1467-fold decrease in K2e between the affected and normal cultures. Although in vitro keratin expression did not precisely simulate in vivo, the morphology of the normal and the affected epidermis was largely preserved; thus, this culture system may provide an alternative to in vivo investigations for cutaneous research involving cornification.