Anti-TLR5 polyclonal antibody

A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3-5 x coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60 x per gene). The diversity in conserved CRP and CR was similar to the diversity in conserved and modern CRP, representing 76.4% and 70.9% of its variants, respectively. Sixty-eight (54.4 %) polymorphisms in the five TLR genes were shared by the two breeds, whereas 38 (30.4 %) were specific to the production herd of CRP; 4 (3.2 %) were specific to the broad CRP population; 7 (5.6 %) were present in both conserved populations; 5 (4.0 %) were present solely for the conserved CRP; and 3 (2.4 %) were restricted to CR. Consequently, gene pool erosion related to intensive breeding did not occur in Czech Simmental cattle. Similarly, no considerable consequences were found from known bottlenecks in the history of Czech Red cattle. On the other hand, the distinctness of the conserved populations and their potential for resistance breeding were only moderate. This relationship might be transferable to other non-abundant historical cattle breeds that are conserved as genetic resources. The estimates of polymorphism impact using Variant Effect Predictor and SIFT software tools allowed for the identification of candidate single-nucleotide polymorphisms (SNPs) for association studies related to infection resistance and targeted breeding. Knowledge of TLR-gene diversity present in Czech Simmental populations may aid in the potential transfer of variant characteristics from other breeds.

Background: Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. Objective: We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. Methods: A truncated version of flagellin (NtCF1g) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1 specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. Results: Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell stimulatory and reduced IgE-binding capacity and induced murine Bet v 1 specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCF1g fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. Conclusion: Bet v 1 flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics.