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Rezultaty
We have demonstrated that CSFV genes Erns, E2 and NS3-4B are targets for the cellular immune system and further analysis is needed with cytotoxic T-cell (CTL) assays. In order to obtain stable clonally cell lines expressing the genes of interest, E2, Erns, NS3-4B, which are based in the vector pEVHis, are transfected into MAX cells. Since E2 appeared to be an important target for CTLs we are interested which domain will be recognised within this E2 domain. In order to determine which domain in the E2 domain is recognised by the CTL, also a clonally cell line expressing E2 with a deleted A domain was obtained. The obtained results indicated that the BC domain of E2 most likely contains important CTL epitopes. Unfortunately, a clonally cell line expressing E2 with a deleted BC domain could not be made. Further experiments will be needed to elucidate this.
Vaccine candidates Flc9 (BVDVII E2 exchanged in C-strain), Flc11 (BVDVII Erns exchanged in C-strain) and vaccine strains C-strain (Flc2) and a trans-complemented Erns deletion mutant (Flc23) derived from the C-strain were studied in animal experiments. PBMCs of these vaccinated animals were used in CTL assays. It was concluded that the Erns, E2 and NS3-4B* proteins were targets for the cellular immune system. The observed cellular immune response was short in time and differences between the vaccine strains were relatively small.
The identification of MHC class II epitopes in viral proteins is an essential point for the investigation of the humoral immuneresponse after a viral infection. In addition, the knowledge of defined T-cell epitopes is important for the development of novel viral vaccines. Overlapping 15mer peptides derived from the protein sequence of CSFV strain Glentorf were synthesized. For the generation of CSFV -specific T lymphocytes, three dfd-haplotype pigs were infected with CSFV-strain Glentorf .Plasma and blood samples were collected before infection and weekly post-infection. CSFV -specific T lymphocytes could be detected by lympho-proliferation assay using PBMC from CSFV-infected animals and CSFV strain Glentorf for the in vitro re-stimulation.
The experiments performed in this project have shown that both CD4+CD8- T-helper cells and CD4-CD8+ cytolytic T-cells were involved in the CSFV-specific response in vitro. The CSFV-specific T-cell response could be shown by the lymphoproliferation assays based on 3H-thymidine incorporation. In order to identify CSFV-specific T-cell epitopes, the overlapping 15mer synthetic peptides derived from CSFV strain Glentorf were applied in these proliferation assays. The 3H-thymidine incorporation assays identified 26 peptides distributed throughout the CSFV viral protein. Characterisation of the reactive epitope-specific T -cell fractions by flow cytometry (FCM) analyses using CFSE-staining, together with different combinations of swine- specific monoclonal antibodies showed that mainly CD4+ T-cells were involved.The phenotype of the main responding T -cell population was actually defined as CD4+CD8a+CD8a-CD45RC-MHCII+.
These analyses with CFSE-stained PBMC, together with identification of the surface antigen expression gave dear hints concerning the possible MHC-restriction of the responding T -cell subset. Therefore, these assays might replace blocking experiments employing monoclonal antibodies against CD4, CD8 MHCI and MHCII molecules. For the determination of the frequency of CSFV -specific T lymphocytes in PBMC from CSFV-infected animals, ELISPOT assays were established to detect interferon-a (IFN-alpha) and Interleukin 4 (IL-4) producing T cells. From these ELISPOT assays, the average frequency of IFN-alpha IL-4 secreting CSFV -specific T lymphocytes in PBMC preparations was calculated as between 1xl0-3 and 1xl0-4.
For a fine analysis the identified CSFV -specific T -cell epitopes were further investigated. They were synthesised for an identification of the amino acids involved in the binding to the MHC-II molecules as "Alanine-Scan-peptides". These "alanine-peptides" were further used in 3H-thymidine incorporation assays, CFSE-assays for the characterisation of the responding T-cell subpopulations and in IFN-y and IL-4 ELIPSOTS for the determination of their activation potency. It could be shown, that an exchange of the amino acids in position 1, 5, 6, 10 and 13 was combined with an reduced stimulation capacity in CSFE-assays, a diminished proliferative response of CSFV -specific T lymphocytes and a reduced frequency of IFN-gamma producing cells.
Cytotoxic T-cells (CTL) provides a key arm of the cellular immune response to viruses. CTLs kill cells infected with viruses and deprive viruses of a site of replication. CTL recognition takes place at the surface of cells and involves recognition of MHC class 1 proteins containing peptides derived from a viral proteins.
An understanding of the nature of peptides bound to MHC class 1 and the viral peptides recognised by CTLs is required for the rational design of vaccines effective against viruses. This project has developed tools to study the natures of peptides bound to porcine MHC class 1 proteins and started to apply this knowledge to a study of proteins encoded by CSFV. Importantly, the genes for pig MHC class 1 proteins have been cloned and sequenced from two lines of inbred pig commonly used to study immune responses to porcine viruses and bioinformatic analysis has identified putative peptide binding grooves.
The genes have been subcloned into vectors that allow recombinant MHC protein to be produced in bacteria and then purified by affinity chromatography. The proteins are now available for refolding studies using overlapping 9mer peptides derived from CSFV in particular, and pig viruses in general. This work paves the way for the design of high through put "in vitor" assays that can identify peptides that bind preferentially to pig MHC class 1 protein within complex pools of peptides.
Natural interferon producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN-a in response to viral and bacterial components, serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN-a after virus infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV) are more receptive to virus infection and produced higher levels of IFN-a than immunologically naive animals. This FcgRII-dependent sensitisation of NIPC was maintained at least for eight months after immunisation. These results demonstrate that NIPC do not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.
These findings are important regarding the role of type I IFN in the limitation of virus production and spreading. Increasing the sensitivity of potent IFN-a producers such as NIPC, in combination with induction of specific immune defences, could be a novel target in the design of vaccines against virus infections.
Vaccination-challenge experiments were performed to study the immunogenic properties of produced candidate vaccine constructs. To determine the kinetics, the CSFV-specific lymphocyte proliferation and the cytotoxic response, mini-pigs (d/d haplotype) were vaccinated with vFlc2 (C-strain) and vFlc9 (C-strain with E2 of BVDV), and challenged with CSFV Brescia. Pereferal blood mononuclear cells (PBMC) of the piglets were tested in CTL assays to determine lysis of various target cells. PBMCs of vFLc2 and vFLc9 immunised piglets could lyse MAX cells that express E2, the BC-domain of E2 and NS3-4B respectively.
Macrophages appeared to be competent target cells for CTL. Flc2 and Flc9 show a CTL response to MAX cells that express E2 and immunogenic properties, but this no difference in response between those two constructs.
Generation of porcine MHC class 1 tetramers specific for CSFV. MHC class 1 tetramers are used to identify peptide specific CTLs. If the peptides are derived from a virus the tetramers can be used in fluorescence activated cell sorting to analyse populations of virus specific T cells isolated following infection and/or vaccination.
The CTLs that bind fluorescent tetramers can be conuter stained with antibodies against lymphocyte surface proteins and the phenotype of the cells recognising infected cells can be determined with precision. This project has produced reagents that will allow this powerful tetramer technology to be used to study pig CTLs produced during infection and or vaccination. The production of tetramesr requires the correct combination of MHC class 1 protien and peptide.
Work in VI (a) above identified a series of 9mer peptides derived from CSFV proteins that are recognised by CTLs of dd pigs infected with CSFV. These petides can now be mixed with recombinant MHC class 1 proteins and a-2 microgobulin (described in V11I-IX) below to make tetramers with the potential to identify CTLs specific for CSFV. This will allow the surface phenotype of CSFV-specific CTLs and the peptides they recognise to be determined with precision.
1. Role of interferon-alpha (IFN-a) responses in the pathogenesis of classical swine fever.
During the acute phase of the viral haemorrhagic disease classical swine fever (CSF) a severe haematological depletion in primary lymphoid organs and depletion of peripheral blood T and B lymphocytes is observed. The onset of these pathological events is before viraemia and independent of leukocyte infection indicating a host-mediated effect possibly through a "cytokine storm".
In this project, we have demonstrated that high serum levels of interferon-a (IFN-a) were found during this phase of CSF, detectable as early as 2 days post infection (p.i.), and reaching maximum levels 3-5 days p.i. This IFN-a response related to the virulence of the virus strain used, with avirulent virus not inducing any detectable serum IFN-a. A progressive depletion of natural interferon producing cells/plasmacytoid dendritic cells, the likely in vivo source of IFN-a, was also induced by the virus infection. An important finding was that the onset of severe lymphopenia was concomitant with the IFN-a responses, and all animals with serum IFN-a had depleted B and T lymphocytes. A statistically significant correlation between lymphocyte depletion and serum IFN-a indicates a relationship between the two events, which would be supported by the known haematological effects of high IFN-a doses in vivo.
An important element within these data is the relationship between the strength of the IFN-a response and the severity of the clinical outcome. This is pertinent not only to CSF, but also to other haemorrhagic fevers such as dengue fever, rift valley fever and South American haemorrhagic fevers. Some of these virus infections induce systemic IFN-a responses, which have been associated with severe disease. The known effect of IFN-a on the haematological system, the IFN receptor knockout studies in mice and the present study demonstrating the correlation between INF-a levels and lymphopenia suggest that an over-production of IFN-a during the acute phase of virus infection can lead to haematological disruption. Keeping the essential role of IFN-a in the innate immune response against virus infections in mind, a balanced regulation of IFN-a responses appears a critical element in the acute phase of certain haemorrhagic fevers, and would have therapeutical potential.
1. IFN-a responses in vaccinated pigs.
The analyses of serum IFN-a responses in naive and vaccinated immune animals would indicate that enhanced sensitisation for IFN-a responses operate in vivo. Our results show that at least 100-2000 less virus in the circulation of immune pigs induced 10-80% of the IFN-a found in the serum of naive pigs early post challenge. However, with highly immune pigs, for example after vaccination with the c-strain no IFN-a responses are detectable. This can be interpreted as a capacity of the vaccine to prevent virus replication. In this sense, the measuring of serum IFN-a after challenge infection of vaccinated pigs can serve as an indication of vaccine efficacy. Ideally, a vaccine would completely prevent the replication of the challenge virus and thus also prevent IFN-a responses.
Antigen presenting cells (APC) with potent immunostimulatory capacities are the critical targets when developing novel vaccines and immunotherapies against infectious diseases and cancer. Although several cell types have the potential for antigen presentation to stimulate T cells, DC are considered to be most potent in this function. In this project we have identified an alternative source of APC for antigen presentation studies with a number of advantages: the fibrocytes.
These cells represent a population of circulating leukocytes reported to be capable of presenting antigen to CD4+ T lymphocytes. We have isolated fiborcates from porcine blood and demonstrated their ability to stimulate CTL responses using a classical swine fever virus (CSFV) model. The isolated fibrocytes displayed the phenotype previously reported for mouse and human Fb, particularly in terms of the surface proteins necessary for antigen presentation - MHC class I and class II, CD80/86. These primary fibrocytes efficiently endocytosed and degraded antigen. In absence of exogenous stimuli, both endocytosis and MHC class II expression were lost when the fibrocytes were passaged and cultured. Treatment of such secondary Fb with IFN-g restored the MHC class II expression. Both the primary and secondary fibrocytes were efficient stimulators of virus-specific CD8+CTL was measured in terms of CD8+ T cell proliferation, IFN-g production and cytotoxic activity. This was noted even at low fibrocyte/T lymphocyte ratios, at which DC were less efficient.
Our results demonstrated that fibrocytes are potent accessory cells for the induction of specific antiviral immunity, particularly with respect to activating virus-specific CD8+ CTL at low Fb/T lymphocyte ratio. Consequently, fibrocytes may prove to be a potent tool for vaccine and immunotherapy research, especially considering their ease of isolation from the blood and adaptability to in vitro expansion (secondary Fb) while retaining APC functionality.
This culture system can be used for the following purposes:
1. Determination of the relative capacity of different vaccines to stimulate cytotoxic T-cell responses in vitro.
2. Identification of genes encoding for proteins, which contain T-cell epitopes.
3. Identification of the T-cell epitopes
4. High-throuput in vitro screening of vaccine candidates.
Novel vaccines and modern delivery systems designed specifically to target dendritic cells (DC) holds promise for improving the immunogenicity of vaccines and modulating the immune response towards the desired direction. Such systems have been established and can be based on synthetic micro- and nanoparticles, virus like particles, virosomes, virus replicating particles or non-particulate system using proteins or nucleic acids linked to a targeting ligand promoting their uptake. The latter of course can also be combined with particulate vaccines. The characterization of the in vitro interactions of such elements with DC permits a "high throughput" screening of delivery systems and vaccine/vaccine components to identify the most potent with respect to their potential to stimulate both innate (DC activation and maturation) as well as adaptive immune responses (Th and Tc responses).
In particular the following has be achieved:
1. A rationalised approach to identify immunogenic viral proteins was established. Due to the access to large volumes of porcine blood, monocyte-derived dendritic cells (MoDC) can be easily generated in vitro in large quantities and used as APC for monitoring T-cell responses and identification of immunogenic proteins or peptides. Although the simple use of blood leukocytes, which already contain APC's can also be used, the employment of MoDC as APC can have an advantage or even be required for certain situations. For example, the ratios of APC can be kept constant and if required high enough to enable efficient T-cell stimulation. Furthermore, MoDC permit controlled antigen delivery, for example when viral vectors, mRNA transfection, micro- or nanoparticle-mediated delivery are used.
In particular the mRNA transfection approach has several major advantages and applications:
(i) It is suitable for MHC class I-restricted CTL epitopes;
(ii) the production of mRNA molecules is easy and quick compared to recombinant proteins,
(iii) in contrast to peptides the approach is not dependent on MHC haplotype,
(iv) MoDCs and fibrocytes can be efficiently transfected with mRNA molecules, whereas gene expression after plasmid DNA transfection is not detectable,
(v) the system is more controllable compared to viral vectors which express many genes with potential to interfere with the experimental system
(vi) mRNA-transfected APCs potentially display the complete spectrum of epitopes relevant to a particular protein, rather than those present on the synthesised peptides. This has the advantage of uncovering previously unknown epitopes, but is particularly valuable in not suffering from restrictions imposed by MHC haplotype.
2. A culture systems for screening of potential immunostimulants suitable as vaccine adjuvants were established.
Using cultures of enriched natural interferon producing cells (NIPC) we have identified vaccines able to induce high levels of IFN-a. These could promise important to improve vaccines in particular for emergency scenarios caused by epidemic outbreaks of virus infections.
The cytokine response of T helper (Th) cells stimulated with antigen can be grouped in a Th1-like or Th2-like response. This has major consequences for the quality of specific effector immune response such as antibody production and cytotoxic activity of T cells.
In this Project we have identified the phenotype of the responding T -helper cell population, determined the frequency of the peptide-specific T -helper cells and the cytokine production pattern (IL-4/ IFN-y). For the characterisation of the CSFV -peptide specific T -cell response PBMC derived from CSFV strain Glentorf-infected dd-haplotype swine were restimulated with synthetic peptides formerly identified in 3H-thymidine proliferation assays as potential T-cell epitopes.
For the restimulation experiments for the more detailed analyses of the T-cell response 14 peptides derived from the non-structural protein region were used. In a first step the responding T -cell subpopulations were characterised in CFSE-proliferation assays by labelling of the in vitro peptide restimulated PBMC-cultures with antibodies against leukocyte differentiation antigens e.g. antibodies against CD4 and CD8. Different patterns of reactive cell subpopulation were obvious: besides peptides inducing nearly only a response of CD4 positive cells either with a weak or strong proliferation, peptides exists which showed a dear stimulatory activity for CD4+ and CD8?+ T lymphocytes. One peptide showed a clear preference for the stimulation of nearly only CD8+ cells. In further experiments for the discrimination of the peptideinduced T -cell response into Thl and Th2-type responses ELISPOT assays for the quantification of the frequency of cytokine producing cells were performed. No correlations could be identified between phenotype of peptide-stimulated cell populations and the frequency of IFN-y/or IL-4 producing cells. Nearly all-possible reactivity patterns were obvious. Peptides with high potency to stimulate IFN-gamma producing ceIls were found as weIl as peptides stimulating both IFN-gamma and IL-4 producing T -ceIls. Also the variation of the frequency of the responding ceIl populations was extremely heterogeneous.
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