Susan Craigmile

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

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.

Quantitative trait loci for variation in immune response to a Foot-and-Mouth Disease virus peptide

BackgroundInfectious disease of livestock continues to be a cause of substantial economic loss and has adverse welfare consequences in both the developing and developed world. New solutions to control disease are needed and research focused on the genetic loci determining variation in immune-related traits has the potential to deliver solutions. However, identifying selectable markers and the causal genes involved in disease resistance and vaccine response is not straightforward. The aims of this study were to locate regions of the bovine genome that control the immune response post immunisation. 195 F2 and backcross Holstein Charolais cattle were immunised with a 40-mer peptide derived from foot-and-mouth disease virus (FMDV). T cell and antibody (IgG1 and IgG2) responses were measured at several time points post immunisation. All experimental animals (F0, F1 and F2, n = 982) were genotyped with 165 microsatellite markers for the genome scan.ResultsConsiderable variability in the immune responses across time was observed and sire, dam and age had significant effects on responses at specific time points. There were significant correlations within traits across time, and between IgG1 and IgG2 traits, also some weak correlations were detected between T cell and IgG2 responses. The whole genome scan detected 77 quantitative trait loci (QTL), on 22 chromosomes, including clusters of QTL on BTA 4, 5, 6, 20, 23 and 25. Two QTL reached 5% genome wide significance (on BTA 6 and 24) and one on BTA 20 reached 1% genome wide significance.ConclusionsA proportion of the variance in the T cell and antibody response post immunisation with an FDMV peptide has a genetic component. Even though the antigen was relatively simple, the humoral and cell mediated responses were clearly under complex genetic control, with the majority of QTL located outside the MHC locus. The results suggest that there may be specific genes or loci that impact on variation in both the primary and secondary immune responses, whereas other loci may be specifically important for early or later phases of the immune response. Future fine mapping of the QTL clusters identified has the potential to reveal the causal variations underlying the variation in immune response observed.

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 24u2003h. 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.

Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

CattleDRA andDRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfectedDRB gene clearly showed showed that it wasDRB3 alleleDRB3*0101, which corresponds to the 1D-IEF-determined alleleDRBF3. 1D-IEF analysis of the transfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigenspecific CD4+ T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3+ heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design.

CHARACTERIZATION OF CATTLE CDNA SEQUENCES FROM TWO DQA LOCI

The class II region of the major histocompatibility complex (MHC) of cattle encodes antigen-presenting molecules of two isotypes, DR and DQ. These highly polymorphic cell-surface glycoproteins bind peptide fragments from mainly exogenous antigens and present them to CD4 T cells to initiate an immune response. Each DR or DQ molecule can bind a range of antigenic peptides, defined by the shape and charge properties of the antigen binding cleft (Brown et al. 1993). Thus, the expression of a wide range of different class II molecules could increase the range of antigens presented to the immune system. Each class II haplotype expresses a single DR molecule, encoded by the DRA and DRB3 genes, but one or more DQ products because of the duplication which occurs in about half of the common class II haplotypes (Andersson and Rask 1988). TheDQ locus is duplicated in primates, but the DQA2andDQB2genes are transcriptionally silent (Kappes and Strominger 1988). In contrast, the DQB genes on duplicated cattle haplotypes are expressed (Bissumbhar et al. 1994; Xu et al. 1994; Marello et al. 1995). In order to correlate class II gene expression and polymorphism with immune function, we are cloning and transfecting the class II genes expressed by a pair of immunologically characterized Holstein-Friesian cattle. The animals (numbers 10795 and 10814) have well-characterized responses to immunization with a model peptide antigen, FMDV15, derived from foot-and-mouth-disease virus (Glass et al. 1991, 1992; Glass and Millar 1994), and had been extensively typed as part of the fifth BoLA workshop (Davies et al. 1994). TheMHC types carried by the animals were: 10795 BoLA-A11,DRB3*0102, DQA1A, DQB1 (class II haplotypeDH24A); BoLA-A36, DRB3*1201, DQA12, DQB12 (class II haplotypeDH8A). 10814 BoLA-A11,DRB3*0102, DQA1A, DQB1 (class II haplotypeDH24A); BoLA-A32, DRB3.2*15, DQA1E, DQB1 (class II haplotypeDH15B). Presentation of the FMDV15 antigen by mouse L cells transfected with theDRA-DRB3gene pair from the shared DH24A haplotype has been described previously (Fraser et al. 1996). Here we report the DQA sequences expressed by these animals, determined from polymerase chain reaction (PCR)-amplified cDNA clones. The lack of extensive DNA sequence data for the cattle DQAgenes led us to use the available sequences from cattle and sheep to design primers which could amplify fulllength DQA genes from cattle cDNA preparations. To improve the chances of amplifying all possible DQA sequences, one forward and two reverse primers were designed. All three primers contained degenerate bases to take into account positions which were polymorphic in the DQA sequences used. The forward primer DQAFWD (59-CCA CCT TGA GAA SAG GAT GRT CCT G-39) annealed at the 5 9 end of theDQA gene and included the start codon (underlined). The reverse primers DQAREV1 (59-ACT TKG SCA GAA AMT AGY TCT AGG-39) and DQAREV2 (59-TGA GAT GAT AYA GCA AYC TTA AGT CC-39) annealed in the 3 9 untranslated region, approximately 70 and 140 base pairs (bp), respectively, beyond the termination codon. Full-length DQA sequences were amplified from firststrand cDNA from both animals using a high-fidelity PCR system (Expand High Fidelity, Boehringer Mannheim, Lewes, UK) to reduce the frequency of PCR artefacts. Amplifications using the DQAFWD-DQAREV1 and DQAFWD-DQAREV2 primer pairs produced clean products of the expected sizes (880 and 950 bp, respectively) from animal 10795, but only the larger product was obtained from animal 10814 (Fig. 1). Despite several experiments using different RNA and cDNA preparations, The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases and have been assigned the accession numbers Y07819, Y07820, and Y07898

Molecular cloning and sequencing of a cattle DRA cDNA.

To date there is full-length sequence information for only two cattle MHC class 11 genes, the DQB gene on the genomac clone Y1 (Groenen et al. 1990) and the DRB3 cDNA clone (Burke et al. 1991). It is known that most sequence polymorphisms are located m the peptide binding domain, especially in exon 2 of the chain. However, proper correlanon of immune fanctaon with sequence polymorphimn necessarily involves studying the complete expressed molecule. In thas commumcation we report the cloning and sequencing of the entire coding region of a cattle DRA gene. Peripheral blood monneytas were purified from whole blood of Fries~an (Bos taurus) cattle with class 1I types DRBF 6,3. Reverse mmscriptmn-polymerase chain reaction (RT-PCR) was carried out on total RNA, using oligo-dT and primers derived from the pubhshed full-length sheep DRA cDNA sequence (Fabb et al 1993), m a reacuon volume of 50 gl comprising 10 mM Trts-Hcl pH 8.3, 50 mM KC1, 100 pg/ml gelatin, 2 mM MgCh, and 1 umt Taq polymerase (Boehrmger Mannhetm~ Lewes, UK). Amplificataon was primed wath 25pmolas of forward (5-CACCAAAGAAGAAAATGGCC-Y) and reverse (5-TGAGACCCACTI~AAGTITACTGTATTC-Y) primers and conststed of 20 cycles of 94 ° C for ] man, 55 ° C for 2 min, and 72 ° C for 3 mm, followed by a 7 mm extension at 72 ° C. ~ products were cloned into the TA vector pCR 11 (Invitrogen, San Dtego, CA) and sequenced in both directions, using Sequenase Vermon 2 0 enzyme (US Biochemacah, Cleveland, OH). We present here (Fig. 1) the sequence of a cailie DRA cDNA. The 1195 base pairs (bp) sequence contains a 762 bp reDon coding for a 253 residue polypeptade. Companr, on with the sequences of exons 2, 3, and 4 from the truncated genomic clone W3 (van der Pcel et al. 1990) shows that the two genes are idenUcal over the regton aligned, thus confirming the idenUty of the DRA cDNA clone and transcription of the W3-encoded gone. The degree of identaty between the translated cattle DRo~ chain sequence and the human DRa sequence ts 80%, and conserved residues of functional sigmficance are highhghted in Figure 1. Interestingly, these residues include E88 and K l l l wlmch may be important m the observed dimerisanon of I-ILA DRI ct/~ he~erodimers (Brown et al. 1993). However, It IS unclear whether this dimerisafion occurs m wvo or if It is ftmclaonally significant.

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.

Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice.

Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose significant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identification of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele. The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed.

Accelerated screening of cDNA libraries using the polymerase chain reaction and Southern blotting

cDNA libraries are normally constructed in either phage or plasmid vectors and screened for sequences of interest using antibodies or, more commonly, nucleic acid probes. To clone a sequence of interest from a library generally involves at least three rounds of hybridization with 32P-labeled probes. This approach is highly labor intensive, and no information about the size of the hybridizing insert is obtained until the clones have been purified and the insert DNA analyzed by restriction enzyme digestion. We report on a rapid screening protocol for libraries constructed in bacteriophage lambda vectors involving polymerase chain reaction amplification of the insert from hybridizing phage plaques and on its analysis by agarose gel electrophoresis and Southern blotting. This can take place after only one round of conventional screening, and phage from a large number of positively hybridizing plaques can be analyzed by a one-tube reaction.