BoLA-DR peptide binding pockets are fundamental for foot-and-mouth disease virus vaccine design in cattle.

This large study investigates the associations between bovine major histocompatibility complex DRB3 alleles and their binding pockets with the immune response to a 40-mer peptide derived from foot-and-mouth disease virus (FMDV) VP1. A crossbred (Charolais and Holstein) cattle population (n=197) was immunised with the FMDV peptide and specific IgG1 and IgG2 responses were measured. Eighteen different DRB3 alleles were detected in this population, with several exhibiting highly significant associations with antibody response. Allele DRB3*1601 was correlated with relatively low IgG1 and IgG2 responses (p<0.001), whereas DRB3*1001 was associated with relatively high IgG1 and IgG2 responses (p<0.001). In contrast the allele *0901 which ranked highest for IgG1 response, only came 14th for IgG2 response. The amino acids at several positions within the peptide binding cleft of the DR molecule showed significant associations (p<0.001) with the level of antibody response. Further analysis showed that specific residues within binding pockets are likely to be crucial to vaccine design. In particular, polymorphisms at position beta70 in pocket 4 were strongly linked to the magnitude of response and highly significant associations were found for position beta57 in pocket 9 and position beta56 in pocket 10. Glutamic acid at position beta70 was associated with low FMDV peptide specific IgG1 and IgG2 response, whereas arginine at beta70 was associated (p<0.001) with a high FMDV peptide specific IgG1 and IgG2 levels. The data indicates that the amino acids within the binding pockets of the DRB3 alleles are critical for determining the degree of immune response and in addition may affect the ratio of IgG1/IgG2, which in turn will influence the efficacy of the peptide to induce protective immunity.

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

Diminished IL-2 responses and alteration of CD2 expression on CD8+ T cells are associated with a lack of cytotoxic T cell responses during Theileria annulata infection.

Theileria annulata is a tick-borne protozoan parasite which causes the disease bovine tropical theileriosis. In immunized or drug-treated animals, the pathogenic macroschizont stage of the parasite is destroyed by MHC class I-restricted cytotoxic T lymphocytes (CTL). Here we show that although CD8+ T cells increase greatly in number and display activation markers during an acute infection, they exhibit no killing of infected cells. During the ineffectual response, efferent lymph cells' ability to proliferate to IL-2 drops, coinciding with loss of MoAb binding to CD2 by CD8+ cells. When animals were treated with the anti-parasite drug 'Butalex', IL-2 responses, anti-CD2 antibody binding by CD8+ cells and strong CTL activity were restored within 24 h. The initial activation of CD4+ T cells by parasite-infected cells altering the IL-2 production in the draining lymph node is the likely cause of the failure of CTL responses.