E. J. Hensen

Vaccine-Induced Immunopathology during Bovine Respiratory Syncytial Virus Infection: Exploring the Parameters of Pathogenesis

ABSTRACT The bovine and human respiratory syncytial viruses cause severe lower respiratory tract infections. Effective vaccines against the respiratory syncytial viruses have been lacking since vaccine failures in the 1960s and 1970s. In this report, we describe a bovine respiratory syncytial virus (bRSV) challenge model in which both classical bRSV respiratory infection and vaccine-enhanced immune pathology were reproduced. The classical, formalin-inactivated (FI) bRSV vaccine that has been associated with vaccine failure was efficient in inducing high antibody titers and reducing viral loads but also primed calves for a far more serious enhanced respiratory disease after a bRSV challenge, thereby mimicking the enhanced clinical situation in FI human RSV (hRSV)-immunized and hRSV-infected infants in the 1960s. We show that immunization with FI-bRSV mainly primes a Th2-like inflammatory response that is characterized by a significant eosinophilic influx in the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies. The current model may be useful in the evaluation of new bRSV candidate vaccines for potency and safety.

A Cartilage-Mimicking T-Cell Epitope on a 65K Mycobacterial Heat-Shock Protein: Adjuvant Arthritis as a Model for Human Rheumatoid Arthritis

The way the immune system evolved has been commonly thought to have been influenced during evolution through selective pressure exerted by microbial invaders. So, immune recognition and the subsequent immune response have become significant means of the host to combat the attack of exogenous invaders. Since, however, the exogenous microbial world presents itself with a wealth of antigenic variety on each single organism, immune recognition can be selective at the antigen level. Teleologically, an unselected response would be a very inefficient maneuver inevitably leading to jamming of the system. Moreover, one could envisage more pertinent reasons for being selective, among them that responding to certain antigens might jeopardize the maintenance of self integrity. This means that a response directed to such antigens might cause uncontrollable disturbances in the balance of elements that interact in the immunological network and might lead to the ultimate development of immunopathology. This could happen when the exogenous stimulator antigen bears a resemblance to self molecules, a situation called antigenic mimicry.

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

Abstract New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC–peptide–TCR interactions either at the level of peptide–MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptldes one must be able to assess peptide–MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptldes. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.BI molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptldes can be assessed while employing detection of blotlnylated marker peptides by chemllumlnescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptldes into good binders. These results are relevant to the design of competitor peptldes in the treatment of experimental autoimmune diseases.

A biochemical characterization of feline MHC products: Unusually high expression of class 11 antigens on peripheral blood lymphocytes

The polymorphism of feline MHC antigens was examined using biochemical methods. The following observations were made: (1) feline class I and II antigens are polymorphic. Their biochemical features were established using rabbit and mouse reagents directed against human MHC products; they resemble those observed for other mammalian species; (2) the expression of class II antigens in unstimulated cat peripheral blood lymphocytes (PBLs) appears to be unusually high. Cat PBLs express far more class II than class I antigens, whereas in human Epstein-Barr virus-transformed lines, which are known to express relatively large amounts of class II antigens, the situation is reversed.

Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein.

Abstract Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.

Biochemically defined polymorphism of bovine MHC class II antigens

The use of biochemical methods has proved to be worthwile for typing and characterization of the human major histocompatibility complex (MHC) antigens (Charron and McDevitt 1980, Shackelford and Strominger 1980, Bontrop et al. 1986, Neefjes et al. 1986). Recently, we reported on the comparison of bovine lymphocyte antigens (BoLA) class I serotyping and one-dimensional isoelectric focusing (1D-IEF) (Joosten et al. 1988). The 1D-IEF data improved and extended the information obtained by classical serology. Concerning BoLA class II typing, all methods used thus far have their drawbacks. The employment of serotyping techniques is hampered by the problem of producing the appropriate antisera. Mixed lymphocyte reaction (MLR) studies have not produced an unambiguous classification. So far, restriction fragment length polymorphism (RFLP) studies using human cDNA probes have been useful, establishing an extensive DR-like and DQ-like polymorphism (Andersson et al. 1986a, 1986b, Sigurdardottir et al. 1988). Here we describe the use of a 1D-IEF technique (Neefjes et al. 1986) for the characterization of BoLA class II antigens. Peripheral blood mononuclear cells (PBM) of 98 cattle (59 British Friesians and 39 Dutch Friesians) were metabolically labeled with 35S methionine, without prior stimulation. Triton X-114 lysates of these cells were incubated with rabbit anti-human MHC class II sera. The immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 1D-IEF (after neuraminidase treatment). The possibility to precipitate bovine MHC class II products using human anti-DR sera was previously shown by Hoang-Xuan and co-workers (1982). The anti-DR serum we used precipitated bovine products, with an apparent relative mass (Mr) of 29000-32000 on a 10% SDSPAGE gel (Fig. 1). A sample of nonstimulated human

The mycobacterial 65 kD heat-shock protein and autoimmune arthritis

SummaryArthritis — induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis. Recent findings from the analysis of immunological reactivity to the 65-kD in rheumatoid arthritis patients seem to support such a role for the mycobacterial 65-kD heat-shock protein in human disease.

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection.

Abstract Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01+ PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1β, IL-6, IL-8, IL-10, IL-18 and IFN-γ mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.

ELISPOT and intracellular cytokine staining: Novel assays for quantifying T cell responses in the chicken.

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-gamma) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-gamma production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-gamma production by CD4+ and CD8+ cells.

Bovine MHC class II restriction fragment length polymorphism linked to expressed polymorphism.

The present study established the association between bovine MHC class II RFLPs and polymorphism at the product level as defined by IEF of immunoprecipitates. To assess the degree of association between RFLP and expressed polymorphism as revealed by IEF analysis, two distant breeds (Swedish Red and White and Dutch Friesian) were used