In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Bovine neonate is deficient in innate immunity at birth.

With an objective to understand acquisition of innate immunity in bovine neonates, we analyzed perinatal expression of cytokine, adhesion molecule and complement component genes involved in innate and adaptive immune functions. Statistically robust transcriptomic analysis of 27 cytokines showed low IL1B, IL2 and IL7 but high IL23, TGFB1 and TGFB2 expression in bovine neonates post-birth. Unlike mice and humans, no TH2 polarizing cytokine expression occurs in bovine neonates. Further, TH17 and Treg differentiation in bovine neonates may differ from other species like mice and humans. Decreased IL7, IL23R, CXCR3 and increased TGFB1 and TGFB2 expression provides an immunosuppressive environment in the bovine neonate at birth. Transcriptomic analysis of 31 adhesion molecules showed rapid increase in ITGAL expression within a week post-birth in bovine neonates that permits acquisition of innate cytotoxic functions by granulocytes (antibody-mediated), cytotoxic T and NK cells. However, innate immune functions involving phagocytosis and platelet aggregation are deficient in bovine neonates at birth. Of twenty-seven, 18 complement component genes show no significant differential gene expression in neonates post-birth. But low expression of C1QA, C1QB, CQC, C1R and C2 compromises classical and lectin complement pathways mediated lytic function in bovine neonates. The complement-mediated cytotoxic functions, however, normalize between days 7 and 28 post-birth. To conclude, bovine neonate is immunosuppressed and deficient in innate immune competence at birth. Such differences with regard to global innate immune deficiency and lack of TH2 polarization in bovine neonates have profound implications for designing vaccines to prevent neonatal infections. To conclude, species-specific unique characteristics of developing innate and adaptive immune system need to be taken into consideration while designing new immunization strategies to prevent neonatal mortality from infections.

Bovine T cell receptors and γδ WC1 co-receptor transcriptome analysis during the first month of life.

Here we evaluated neonatal transcription of α, ß, γ and δ TCR and the γδ T cell co-receptor family WC1 in peripheral blood mononuclear cells. A previous report showed a rapid and global shift in transcription of immunoglobulin genes in neonatal calves during the first month after birth but this was not found here for the T cell genes. Transcription frequency of genes within TRAV subgroups correlated with the number of members, indicating a stochastic choice. In contrast, of the approximately 60 TRDV genes those in two of eleven TRDV1 clades and TRDVb3 were transcribed significantly more than the others while those in only one TRBV subgroup were. Transcription of genes in the TRGV5-containing cassette predominated among TRGV genes as a result of their exclusive usage by the WC1+ γδ T cells with a preference for transcription of two of four TRGV genes in that cassette. Finally, we report no large differences in transcription frequencies among the 13 WC1 genes.

Preferential expression of IGHV and IGHD encoding antibodies with exceptionally long CDR3H and a rapid global shift in transcriptome characterizes development of bovine neonatal immunity.

With an objective to understand natural development of bovine neonatal immunity, we analyzed 18 RNA-seq libraries from peripheral blood lymphocytes of three neonatal calves pre- (day 0) and post-colostrum (7, 14 and 28) uptake as compared to their dams. A significant global shift in neonatal transcriptome occurs within first week post-birth, in contrast to dams, with an upregulation of 717 genes. Global pathway analysis of the transcriptome revealed 110 differentially expressed immune-related genes, such as, complement, MHCII, chemokine receptors, defensins and cytokines, at birth. The signaling molecules (LAX1, BLK) and transcription factors (GATA3, FOXP3) are expressed at high levels. High expression of GATA3 transcription factor at birth seems to skew the neonatal immune response towards TH2 type. The high levels of T-cell signaling molecules, CD3G and CD3D, at birth are important in neonatal T cell development. Unlike adults, IGKC expression is high in the neonates where IGKV12 is preferentially expressed at birth. But IGLC is predominant in both neonates and adult where IGLV3.4 is preferentially expressed in B cells at birth. Both IGHM and IGHD are expressed at birth and IGHM achieves adult levels by day 7. This is followed by IGHA and IGHG expression 14-28 days post-birth. Importantly, preferential expression of IGHV1S1(BF4E9) and longest IGHD2(DH2) genes that encode immunoglobulin with exceptionally long CDR3H at birth indicates their critical role, as B cell antigen receptor, in the B cell development via idiotype-anti-idiotype interactions. The transcriptome signatures described here permit assessment bovine neonatal immunocompetence. Bovine neonates acquire innate and IgM-mediated humoral immunocompetence within first week post-birth.

Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.

We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity.

Enhanced bovine herpesvirus type 1 neutralization by multimerized single-chain variable antibody fragments regardless of differential glycosylation.

Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.

Organization and genomic complexity of bovine lambda-light chain gene locus.

Complete characterization and physical mapping of bovine lambda (lambda) light chain locus, spanning 412kbp, on chromosome 17, has revealed twenty-five V(lambda) genes, seventeen being functional, organized in three sub-clusters 23.7kbp 5' of the J(lambda)-C(lambda) units. Three V(lambda) sub-clusters are separated by two large introns of 126.8 and 138.3kbp. The predominantly expressed V(lambda)1 genes are present in the two 5' sub-clusters, while J(lambda)-proximal V(lambda) sub-cluster comprises rarely expressed V(lambda)2 and V(lambda)3 genes. The preferential expression of V(lambda)1 genes in the bovine immunoglobulin repertoire is influenced by the composition of recombination signal sequences (RSS). Of the J(lambda)-C(lambda) cluster, it is mainly J(lambda)3-C(lambda)3 unit that is expressed in reading frame 2, though J(lambda)2 and J(lambda)3 have identical RSS. The predominant expression of J(lambda)3-C(lambda)3 genes over J(lambda)2-C(lambda)2 is likely due to endogenous counter selection for J(lambda)2 encoded CDR3 and framework 4 regions. Differences in the genomic complexity of V(lambda) genes in Hereford and Holstein cattle are due to polymorphism at the lambda-light chain gene locus. Despite more potential germline encoded combinatorial diversity, restricted V(lambda)1-J(lambda)3-C(lambda)3 recombinations encode the most lambda-light chain repertoire in cattle.