Bovine IFN Research Articles

A slow release formulation for recombinant bovine interferon αI-1

Recombinant bovine interferon- α I 1 (rBoIFN-α) has known antiviral and immunomodulatory effects which have been exploited to reduce clinical disease in a number of clinical situations including bovine respiratory diseases. A slow release rBoIFN-α formulation may be of value to reduce bovine respiratory disease under field conditions by extending the period of protection, and hence improving the prophylactic benefits of rBoIFN-α. In this report, we describe a formulation of rBoIFN-α in sesame oil containing calcium stearate which can successfully sustain the release of rBoIFN-α over an 8-day period. Recombinant bovine IFN-α could be measured in serum for 8 days following treatment with an initial burst of release 6 h after injection. After a single subcutaneous depot injection of 50 mg and 100 mg of rBoIFN-α, initial serum levels reached 12–15 ng/ml and 25 ng/ml respectively. Correlating with this burst of release, there was a decrease in the number of circulating CD4 −CD8 − γδ + T lymphocytes, and a slight neutropenia. No alterations in other cell phenotypes tested (CD4, CD8, CD2, CD6, B cells, monocytes or MHC class II) were observed, nor were there changes in lymphokine activated killer (LAK), natural killer (NK) cell activity, or oxygen radical formation (assessed by reduction of nitroblue tetrazolium). However, despite the rapid and short-lived burst of rBoIFN-α, levels of 2–5 oligoadenylate (2–5 A) synthetase remained elevated for 8 days. The sustained increase of 2–5 A synthetase was not due to the high initial dose released during the burst 6–12 h after injection, since injection of a bioavailable equivalent dose of interferon induced a significant rise in 2–5 A synthetase activity for 4 days only. As 2–5 A synthetase is known to be a correlate of antiviral activity, we propose that this formulation of rBOIFN-α may be one approach to increase the window of protection, leading to more effective prevention of bovine respiratory disease.

Natural and recombinant bovine interferon tau regulate basal and oxytocin-induced secretion of prostaglandins F2 alpha and E2 by epithelial cells and stromal cells in the endometrium.

The effects of bovine interferon tau (IFN tau) and oxytocin on secretion of the prostaglandins PGF2 alpha and PGE2 by epithelial and stromal cells in the endometrium were assessed in two experiments. Endometrial tissues were collected from cyclic cows at Day 15 after oestrus for subsequent isolation of epithelial cells (4 cows) and stromal cells (4 cows). In both experiments, confluent cells were treated with 0, 2, 10 or 50 ng mL-1 natural bovine IFN tau (nbIFN tau) or 0, 0.4, 2, 10, 50 and 250 ng mL-1 recombinant bIFN tau (rbIFN tau). Culture medium was sampled at 24 h. Oxytocin (2.0 x 10(-7) M) or placebo was then added to wells and the medium was sampled 30 and 90 min later. Epithelial cells secreted more PGF2 alpha than stromal cells whereas stromal cells predominantly secreted PGE2. Oxytocin stimulated secretion of PGF2 alpha and PGE2 (P < 0.01) from epithelial cells, but both basal secretion and oxytocin-induced secretion of PGF2 alpha and PGE2 decreased with increasing dose of either nbIFN tau or rbIFN tau (P < 0.01). At comparable doses, rbIFN tau inhibited PGF2 alpha and PGE2 secretion more strongly than did nbIFN tau (either in the absence or the presence of oxytocin). The minimal effective dose of rbIFN tau was 0.4 ng mL-1 and 50% inhibition was obtained with 1 ng mL-1 (0.043 nM). Neither nbIFN tau nor rbIFN tau nor oxytocin altered PGF2 alpha or PGE2 secretion by stromal cells. The results indicate differential prostaglandin responses by the two major endometrial cell types (epithelium and stroma) to regulatory agents such as bIFN tau and oxytocin in cattle. Suppression of prostaglandin secretion by bIFN tau in epithelial cells of endometrial tissue is supportive of an antiluteolytic effect of bIFN tau.

Cloning and structural analysis of two distinct families of ovine interferon-α genes encoding functional class II and trophoblast (oTP) α-inteferons

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II α-interferon (IFN-αII) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-αII gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-αII gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-α genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275–282) than with oIFN-αII gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-αs.

Receptors for Human Interferon α on Bovine Cells: Specificity and Tissue Distribution

Human interferon alpha 1 (HuIFN alpha 1) is known to protect bovine as well as human cells against viral infection. Hence, we investigated the specificity and tissue distribution of receptors for HuIFN alpha 1 on various cells. [35S]HuIFN alpha 1 bound specifically to homogenates of bovine tissues and particularly to bovine liver, but there was also specific binding to spleen, kidney, brain, adrenal gland, lung, thymus, skeletal muscle, heart, mammary gland and testis. There was no difference in the degree of binding of HuIFN alpha to foetal or adult liver. Competitive binding experiments showed that bovine interferon alpha C (BoIFN alpha C) competed with HuIFN alpha 1 for binding to a bovine liver plasma membrane preparation, indicating that these two IFNs bind to the same receptor. An 35S-labelled IFN alpha 1-receptor complex was isolated from bovine liver extracts by SDS/polyacrylamide gel electrophoresis, and shown to have a molecular weight of 153 kDa. Isolation of the bovine IFN alpha receptor would be a feasible approach to the characterization of the HuIFN alpha receptor.

The cysteines in position 1 and 86 of rat interferon-α 1 are indispensable for antiviral activity

The human, bovine, murine and rat interferon (IFN)-α families contain 4 conserved cysteines located at positions 1, 29, 99 and 139 that are involved in disulfide bridges. Rat and murine IFN-α subspecies carry a fifth Cys (Cys-86) which is not conserved in bovine and human IFN-α subspecies except for human IFN-α 1. Changing Cys-86 in rat IFN-α 1, into Ser or Tyr virtually abolished antiviral activity. As shown by others, the substitution of Cys-86 to Ser in human IFN-α 1 had no pronounced effect on activity. This suggests that in contrast to human and bovine IFN-α, Cys-86 in rodent IFN-α plays a crucial role in receptor binding. Changing Cys-1 to Gly in rat IFN-α 1 also destroyed activity, in agreement with results obtained in the human IFN-α 1 system.

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with bhv1 and pi3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.

The action of recombinant bovine interferons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells

Recombinant bovine interferon-α and -γ differ in their action against influenza virus on bovine cells. Bovine IFN-α severely impairs early protein synthesis and replication of influenza virus in bovine cells in contrast to bovine IFN-γ which fails to induce an antiviral state against influenza virus. Otherwise the IFN system seems to function normally in bovine cells since both bovine IFN-α and -γ induce an antiviral state against vesicular stomatitis virus. The establishment of the specific antiviral state against influenza virus correlates with the induction by bovine IFN- a, but not γ, of two cytoplasmic proteins related to the IFN-induced mouse protein Mx involved in the mechanism of resistance of mice to influenza virus infection. This study suggests that bovines possess a system for resistance to influenza virus similar to the mouse Mx system.

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects.

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.