Cell-mediated and humoral immune responses in pigs following primary and challenge-exposure to Lawsonia intracellularis.

To investigate immune responses upon re-infection with Lawsonia intracellularis, local and peripheral humoral and cell-mediated immune responses to primary and challenge inoculations were studied in 22 pigs. Pigs were orally inoculated with virulent L. intracellularis at the age of 5-6 weeks, treated with antibiotics and challenged with a re-inoculation (RE) at the age of 12 weeks. Treatment control (TC) pigs received only the primary inoculation and challenge control (CC) pigs received only the secondary inoculation at 12 weeks of age. Following this regimen, all RE pigs were protected against the re-infection as defined by reduced colonisation and pathology of intestinal mucosa, absence of bacterial shedding and without increase in serum acute phase protein response. In the protected RE pigs, serum IgG responses were variable with both high and low responders. Serum IgA responses were not boosted by the re-inoculation, since identical intestinal IgA responses developed in response to the inoculation in both the susceptible CC pigs and the protected RE pigs. A memory recall cell-mediated immune response developed in RE pigs which was significantly stronger compared to the primary response in age-matched CC pigs as assessed by whole blood IFN-γ assay and by calculation of IFN-γ integrated median fluorescence intensity (iMFI) after flow cytometry. The major IFN-γ producing cells were identified as CD8+ and CD4+CD8+ double positive lymphocytes. The results indicate that cell-mediated immune responses are likely mediators of protective immunity against L. intracellularis, with CD8+ effector cells and CD4+CD8+ double positive memory T cells as main contributors to the antigen-specific IFN-γ production.

Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues.

Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP(Sc)) of the prion protein (PrP(C)) on neuronal cells. These methods depend on antibodies directed against PrP(C) and capable of reacting with PrP(Sc)in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP(Sc), including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Sträussler-Scheinker (GSS) disease PrP(Sc) as well as PrP(Sc) of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP(Sc) blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP(RES)) in situ. Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153-165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153-165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153-165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP(Sc)in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4 reached the same level of reactivity as 1.5D7 and 1.6F4. This shows that 6H4 needs denatured PrP(RES) to reach maximal reactivity, confirming earlier results. As an exception, human PrP(RES) still reacted relatively poorly with 6H4 in PET-blotting, while 1.5D7 and 1.6F4 reacted well with PrP(RES) from most human CJD types. Taken together this implies that the binding epitope of 1.5D7 and 1.6F4 is accessible in the aggregates of undenatured PrP(Sc) (IHC) while the binding site of 6H4 is at least partly inaccessible. In techniques incorporating a denaturing and/or disaggregating step 6H4 showed good binding indicating increased accessibility of the binding site. An exception to this is human samples in PET-blotting suggesting that huPrP(RES) might not be as easily unfolded by denaturation as BSE and scrapie PrP(RES). Also of interest was the ability of 1.5D7 and 1.6F4 to discriminate between two allelic variants of PrP CJD(Sc) (VV vs. MM) in immunohistochemistry as opposed to the normally used antibody 3F4.

Prion-Specific Antibodies Produced in Wild-Type Mice.

Peptide-specific antibodies produced against synthetic peptides are of high value in probing protein structure and function, especially when working with challenging proteins, including not readily available, non-immunogenic, toxic, and/or pathogenic proteins. Here, we present a straightforward method for production of mouse monoclonal antibodies (MAbs) against peptides representing two sites of interest in the bovine prion protein (boPrP), the causative agent of bovine spongiform encephalopathy ("mad cow disease") and new variant Creutzfeldt-Jakob's disease (CJD) in humans, as well as a thorough characterization of their reactivity with a range of normal and pathogenic (misfolded) prion proteins. It is demonstrated that immunization of wild-type mice with ovalbumin-conjugated peptides formulated with Freund's adjuvant induces a good immune response, including high levels of specific anti-peptide antibodies, even against peptides very homologous to murine protein sequences. In general, using the strategies described here for selecting, synthesizing, and conjugating peptides and immunizing 4-5 mice with 2-3 different peptides, high-titered antibodies reacting with the target protein are routinely obtained with at least one of the peptides after three to four immunizations with incomplete Freund's adjuvant.

Vaccination of pigs with attenuated Lawsonia intracellularis induced acute phase protein responses and primed cell-mediated immunity without reduction in bacterial shedding after challenge.

BACKGROUND: Lawsonia intracellularis causes porcine proliferative enteropathy and is one of the most economically important diseases in modern pig production worldwide. The Enterisol Ileitis vaccine have been shown to reduce clinical disease and to increase weight gain, however, while the natural infection with L. intracellularis can provide complete protection against re-infection, this has not been achieved by this vaccine. We therefore undertook a detailed characterization of immune responses to L. intracellularis infection in vaccinated pigs (VAC) compared to previously infected pigs (RE) in order to pinpoint immunological determinants of protection. RESULTS: The VAC pigs shed L. intracellularis to the same extent as non-vaccinated pigs after challenge, however less L. intracellularis in ileum and lymph nodes was seen post mortem. In the RE group, challenge did not lead to L. intracellularis shedding and no challenge bacteria were found post mortem. In both VAC and RE the acute phase haptoglobin response was diminished and L. intracellularis specific IgG responses were delayed and reduced compared to non-vaccinated pigs. On the other hand L. intracellularis specific IFN-γ responses tended to develop faster in the VAC group compared to controls. CONCLUSION: Although vaccinated and non-vaccinated pigs shed L. intracellularis at similar levels after challenge, a lower number of intestinal L. intracellularis was observed in the vaccinated pigs at post mortem inspection. This might be due to the observed faster CMI responses upon challenge in vaccinated pigs. Complete protection against infection without L. intracellularis shedding, however, was only seen after a previous infection resulting in IFN-γ production predominantly by CD8(+) and CD4(+) CD8(+) cells. Improved protective vaccines against L. intracellularis should therefore target stimulation of these T cell subsets.

Primary infection protects pigs against re-infection with Lawsonia intracellularis in experimental challenge studies.

In two separate trials pigs were experimentally infected with Lawsonia intracellularis at 5-6 weeks of age followed by antibiotic treatment and resolution of the primary infection and then re-inoculated at 12-13 weeks of age. A treatment-control group of pigs received the primary infection and antibiotic treatment only, and served as control for the antibiotic treatment of the primary infection. A challenge-control group of pigs received the second inoculation dose only at 12-13 weeks of age to control infectivity of the challenge-dose and susceptibility of pigs to L. intracellularis at this age. Pigs were monitored for shedding of L. intracellularis in faeces by PCR, and for the development of antibodies and responses of acute phase proteins in serum. The presence of L. intracellularis antigen in the intestinal mucosa was examined in post mortem samples by immunohistochemistry. In both trials primary infected pigs were protected from infection after challenge inoculation as evidenced by absence of faecal shedding of L. intracellularis, lack of changes in acute phase protein concentrations after challenge and with low levels of bacterial antigen in the intestinal mucosa of re-inoculated pigs comparable to that of the treatment-control pigs. In contrast, challenge-control pigs shed L. intracellularis in faeces, had L. intracellularis antigen extensively present within all layers of the intestinal mucosa and developed a significant acute phase protein response in serum after the experimental infection. The acute phase protein response to L. intracellularis infection was detected as an increased rise in the serum concentrations of C-reactive protein and haptoglobin from day-6 post infection, and increased serum concentrations of haptoglobin were generally seen 2-3 weeks after inoculation both at 5-6 and 12-13 weeks of age. In conclusion substantial protection against L. intracellularis infection was found in the re-inoculated pigs in contrast to the development of infection in age-matched control pigs. The acute phase protein responses reflected both the observed protection against L. intracellularis infection upon secondary challenge and that increased resistance to the infection develops with age.

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge.

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.

Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro.

One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrP(Sc)) of the cellular prion protein (PrP(C)) primarily in the central nervous system. The synthetic prion peptide PrP106-126 shares many characteristics with PrP(Sc) in that it shows PrP(C)-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106-126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106-126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106-126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106-126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106-126 is closely related to its ability to attain secondary structure.