Optimal combinations of acute phase proteins for detecting infectious disease in pigs

Peer reviewedPublisher PD

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26-and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ∼75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immunedeficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals

Pig α₁-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein.

The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1-3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2-5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein

Hepatic expression of pig AGP gene during acute infection.

<p>A: Relative expression levels of pig AGP (left) and pigMAP (right) (mean of controls (CTRL, N = 6) set to 1) at 24 hours after experimental infection with <i>Actinobacillus pleuropneumoniae</i> serotype 6 (Ap6) and serotype 2 (Ap2), respectively, as indicated. Values for all individual animals are shown. Error bars depict SEM. Analysis was done on liver tissue samples by qPCR (see text). P<0.01: **, not significant: NS. B: Relative expression levels (mean of controls (CTRL, N = 2) set to 1) in <i>Staphylococcus aureus</i> liver samples 30 (N = 3), 36 (N = 2) and 48 (N = 2) hours after i.v. infection with the bacterium as determined by qPCR, pig AGP (left), pig MAP (middle) and haptoglobin (right). Controls received sterile isotonic saline and were euthanized at 48 hours. Values for individual animals are shown. Error bars depict SEM.</p

Characterization of pig AGP by SDS PAGE and Western blotting.

<p>A: Left panel: Silver-stained SDS PAGE, from the left: Salted-out pooled pig serum supernatant; purified pig AGP; purified pig AGP after sialidase treatment. Right panel: Western blot with the same samples probed with rabbit anti human AGP (DAKO). Arrow: Position of pig AGP in salted-out serum supernatant. B: Western blot probed with anti human AGP, from the left: Purified pig AGP; purified AGP after sialidase treatment; purified AGP after sialidase and PNGase F treatment; buffer control for PNGase F treatment. C: Western blot of pooled pig serum probed with antiserum (1/500) from mouse immunized with purified pig AGP (see text)(representative example). D: Western blot probed with MAb 1.62, from the left: Pooled pig serum; purified pig AGP; purified pig AGP after sialidase treatment.</p

Serum concentrations of pigAGP during the acute phase response.

<p>Serum concentration of pig AGP (left) and pig haptoglobin (right) at different days post infection after experimental <i>Streptococcus suis</i> (A), <i>Actinobacillus pleuropneumoniae</i> (haptoglobin data not included) (B), and <i>Staphylococcus aureus</i> (C) infection and after aseptic inflammation (D). Note: In the <i>Staphylococcus aureus</i> experiment, only two infected pigs were sampled at 48 hours.</p

Characterization of pig AGP by 2D electrophoresis and 2D blotting.

<p>A: Pig AGP 2-D electrophoresis, influence of sample preparation conditions, from left to right: reduced sample, non-reduced sample, non-denatured sample. Close-up from gels with pH gradient 2.5–5, individual pig serum sample (B16/221). Mw markers: 30,43,67,94 kDa (from bottom). Arrow: Pig AGP isoforms. B: The reaction of MAb 1.62 with non-purified pig PAGP isoforms by 2-D electrophoresis. Individual pig serum sample (Aus), non-reducing, complete gel/blot with IPG pH 2.5–5 in the first dimension, left: silver-stained, right: blot probed with MAb 1.62. Mw markers: 14, 20, 30, 43, 67, 94 kDa (from bottom). Arrow: Pig AGP isoforms C: Close-up of 2-D electrophoresis of two individual pig sera (827 µg/ml (left), 1692 µg/ml (right)), silver-stained (top), blotted and stained by RuBPS (general protein stain)(middle), and the same blot subsequently probed with MAb 1.62 (bottom). Non-reducing, pH gradient 2.5–5. Arrow: Pig AGP isoforms.</p

ELISA quantification of pigAGP.

<p>A: Titration of purified pig AGP, pig AGP standard (Saikin Kagaku Institute Ltd.), and two individual pig sera in competitive MAb 1.62 based ELISA. B: Serum concentrations of pig AGP in newborn piglets and in 1-month old piglets (Landrace, Duroc, Yorkshire crossbreds, N = 31). Bars indicate mean and SEM. C: Serum concentrations of pig AGP in different pig breeds and rearing conditions (individual samples), mean and SEM shown. DD: Duroc (2 months, herd) LL: Landrace (2 months, herd) YY: Yorkshire (2 months, herd) Ossabaw minipigs, Experimental stables (14–16 months of age) Göttingen minipigs, Experimental stables (41–47 months of age) L/Y: Landrace/Yorkshire crossbreds (experimental stables, 8–9 months of age) Conventional herd (5 months, D/L/Y cross bred production pigs) SPF herd (5 months, D/L/Y cross bred production pigs).</p