Bighorn sheep × domestic sheep hybrids survive Mannheimia haemolytica challenge in the absence of vaccination.

Bighorn sheep (BHS, Ovis canadensis) are much more susceptible than domestic sheep (DS, Ovis aries) to pneumonia caused by leukotoxin (Lkt)-producing members of the Family Pasteurellaceae, particularly Mannheimia haemolytica and Bibersteinia trehalosi. Leukotoxin is widely accepted as the critical virulence factor of these bacteria since Lkt-negative mutants do not cause death of BHS. Typically, DS carry Lkt-positive M. haemolytica and/or B. trehalosi as commensal bacteria in their nasopharynx. In contrast, most BHS do not carry Lkt-positive M. haemolytica or B. trehalosi, or carry Lkt-negative strains in their nasopharynx. In previous studies, we demonstrated that unimmunized DS resist M. haemolytica challenge while BHS succumb to it. We hypothesized that Lkt-neutralizing antibodies, induced by Lkt-positive M. haemolytica and/or B. trehalosi innately carried by DS in their nasopharynx, render them less susceptible to infection by these bacteria. In this study we developed BHS×DS F1 hybrids by artificial insemination of domestic ewes with BHS semen. F1 hybrids were fertile, and produced F2 hybrids and back-crosses. The F1, F2, and back-crosses were raised together with domestic ewes. All these animals acquired Lkt-positive M. haemolytica and/or B. trehalosi, and developed high titers of Lkt-neutralizing antibodies in the absence of vaccination. Furthermore, all of these animals resisted challenge with lethal dose of M. haemolytica. These results suggest that lack of previous exposure to Lkt is at least partially responsible for fatal pneumonia in BHS when they acquire Lkt-positive M. haemolytica and/or B. trehalosi from DS when the two species commingle.

Mannheimia (Pasteurella) haemolytica leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18.

Previously, we identified bovine CD18 as the receptor for leukotoxin secreted by Mannheimia (Pasteurella) haemolytica. In this study, we constructed bovine-murine CD18 chimeras to locate the leukotoxin binding domain on CD18. Leukotoxin specifically lysed transfectants expressing bovine CD18 fragment encompassing amino acids 1 to 291, indicating that leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18.

Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1.

Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study was to investigate whether EHV-1 interferes with peptide translocation by the transporter associated with antigen processing (TAP) and to identify the proteins responsible. Using an in vitro transport assay, we showed that EHV-1 inhibited transport of peptides by TAP as early as 2 h post-infection (p.i). Complete shutdown of peptide transport was observed by 8 h p.i. Furthermore, pulse-chase experiments revealed that maturation of class I molecules in the endoplasmic reticulum (ER) was delayed in EHV-1-infected cells, which may be due to reduced availability of peptides in the ER as a result of TAP inhibition. Metabolic inhibition studies indicated that an early protein(s) of EHV-1 is responsible for this effect.

Inhibition of TAP by pseudorabies virus is independent of its vhs activity.

Previously we have shown that pseudorabies virus (PrV) down-regulates the expression of porcine MHC class I molecules by interfering with the transporter associated with antigen processing (TAP). During lytic PrV infection, the half-lives of both host and viral mRNA are regulated by the product of virion host shut-off (vhs) gene, UL41. PrV vhs protein induces degradation of cellular mRNA including those encoding class I and TAP. Therefore, further elucidation of specific mechanisms of down-regulation of class I molecules by PrV necessitates construction of a vhs deletion mutant. Two such mutants (vhsDelta1 and vhsDelta2) were generated by homologous recombination between the wild type (wt) PrV Indiana-F strain, and plasmids containing truncated UL41 gene of PrV into which the enhanced green fluorescent protein (EGFP) cassette was inserted. Compared with the wt virus, both the vhs mutants exhibited slower in vitro growth kinetics. The mutants, like the wt virus, inhibited the peptide transport activity of TAP and down-regulated cell surface expression of class I molecules. These findings suggest that, inhibition of TAP activity in PrV-infected cells is due to mechanism(s) specifically directed at class I pathway and not due to the non-specific vhs activity of the virus.

T cell epitope identification for bovine vaccines: an epitope mapping method for BoLA A-11.

T cell responses play an important role in immunity to parasites and other microbial agents of infectious diseases, therefore a number of T cell-directed vaccines are in development. Computer-driven algorithms that facilitate the discovery of T cell epitopes from protein and genome sequences are now being used to accelerate preclinical studies of human vaccines. Similar tools are not yet available for predicting T cell epitopes for animal vaccines, but there may be sufficient data available to begin the process of compiling the algorithms. We describe the construction of a novel mathematical 'matrix' that describes the properties of bovine major histocompatibility complex (BoLA) system antigen (BoLA) A-11 peptide ligands, developed for use with EpiMatrix, an existing T cell epitope-mapping algorithm. An alternative means of developing BoLA matrices, using the pocket profile method, is also discussed. Matrices such as the one described here may be used to develop T cell epitope-mapping tools for cattle and other ruminants. Epitope-mapping algorithms offer a significant advantage over other methods of epitope selection, such as the screening of synthetic overlapping peptides, because high throughput screening can be performed in silico, followed by ex vivo confirmatory studies. Furthermore, using epitope-mapping algorithms, putative T cell epitopes can be derived directly from genomic sequences, allowing researchers to circumvent labor-intensive cloning steps in the genome-to-vaccine discovery pathway.

Induction of cytotoxic T-lymphocytes specific for bovine herpesvirus-1 by DNA immunization.

Cytotoxic T-lymphocytes (CTLs) are critical for the defense against herpesvirus infections, in which cell-to-cell spread occurs earlier than the hematogenous spread. The ability of bovine herpesvirus-1 (BHV-1) to undergo latency, to induce apoptosis of CD4(+) T-lymphocytes, and to down-regulate the expression of major histocompatibility complex (MHC) class I molecules, necessitates the development of immunization strategies that do not involve the live virus. The objective of this study was to evaluate the feasibility of DNA immunization as a means of induction of CTLs against BHV-1. Mice were injected either by intramuscular (IM) or intradermal (ID) route with a Sindbis virus-based plasmid carrying the gene encoding the glycoprotein D (gD) of BHV-1. Splenocytes from the immunized mice were re-stimulated in vitro with gD-transduced syngeneic fibroblasts. The CTLs generated specifically lysed syngeneic targets, either transduced with gD or infected with BHV-1. IM route of inoculation induced a better CTL response when compared to ID route with respect to onset, magnitude and duration of immunity. These results indicate the feasibility of using a plasmid carrying the gene encoding BHV-1 gD as an immunogen to induce CTLs against BHV-1.

Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxin-induced cytolysis.

Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to beta(2) integrins on the surface of bovine leukocytes. beta(2) integrins have a common beta subunit, CD18, that associates with three distinct alpha chains, CD11a, CD11b, and CD11c, to give rise to three different beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three beta(2) integrins, suggesting that the common beta subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.

Effects of virion host shut-off activity of bovine herpesvirus 1 on MHC class I expression.

Previously, we have shown that bovine herpesvirus 1 (BHV-1) down-regulates the expression of major histocompatibility complex class I molecules by interfering with transport of peptides by the transporter associated with antigen processing (TAP). Further studies revealed that BHV-1 down-regulates the expression of mRNA for class I molecules and other cellular proteins. To further elucidate the mechanisms of down-regulation of class I molecules, a virion host shut-off (vhs) deletion mutant was generated. The mutant, like the wildtype (wt) virus, interfered with transport of peptides by the TAP, and down-regulated cell surface expression of class I molecules. However, unlike the wt virus, the mutant did not impair the synthesis of class I molecules. These results indicate that down-regulation of class I molecules by BHV-1 is mediated by vhs activity of the virus, as well as mechanisms specifically directed at the class I pathway. Absence of vhs activity should result in decreased pathogenicity and enhanced immunogenicity of BHV-1 vhs deletion mutant, making it a better vaccine candidate.

Heat shock protein-peptide complexes elicit cytotoxic T-lymphocyte and antibody responses specific for bovine herpesvirus 1.

Epitope-based vaccines offer a promising alternative to modified live vaccines against viruses such as herpesviruses which give rise to latent infections, and induce immunosuppression. The success of this approach depends on the ability to direct the CTL epitopes to the MHC class I antigen presentation pathway. The objective of this study was to evaluate the potential of the heat shock protein gp96 in this regard. A group of BALB/c mice was injected with three murine CTL epitope peptides of bovine herpesvirus 1 (BHV-1) complexed in vitro with bovine gp96 (gp96-peptides). Three other groups were injected with either the peptides alone, gp96 alone, or the peptides complexed with BSA. CTLs from mice immunized with gp96-peptides specifically lysed the peptide-pulsed syngeneic targets, as well as BHV-1-infected targets. CTLs from the other three groups did not lyse these targets. To further evaluate the utility of this approach, groups of BALB/c mice were immunized with gp96 isolated from a syngeneic cell-line transduced with BHV-1 glycoprotein D (BC-gD). Mice immunized with gp96 from BC-gD developed CTLs, as well as Abs specific for BHV-1 gD. Furthermore, in vitro stimulation of naive bovine PBMCs with gp96 from BC-gD resulted in CTLs specific for BHV-1. These results demonstrate the feasibility of using gp96-peptide complexes isolated from cells expressing BHV-1 proteins to induce CTL and Ab responses against BHV-1, without the prior knowledge of the CTL and Ab epitope sequences.

A vhs-like activity of bovine herpesvirus-1.

Bovine herpesvirus 1 (BHV-1) is a major pathogen of cattle, causing significant disease including immunosuppression in infected animals. In vitro, the surface expression of major histocompatibility complex (MHC) class I molecules, crucial for an appropriate anti-viral immune response of the host, is down-regulated by BHV-1 infection. Northern blot analyses revealed that the mRNAs for MHC class I and class II molecules were significantly down-regulated in BHV-1 infected cells, starting as early as 2 h after infection. Furthermore, mRNA expression of the two house keeping genes actin and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was also repressed after infection. This BHV-1 induced effect on cellular metabolism resembled the virion host shutoff (vhs) activity of herpes simplex virus (HSV). Similar to the HSV vhs activity, the putative BHV-1 vhs activity was not abrogated in cells infected in the presence of actinomycin D (ActD) which suggested that no viral gene expression is required for the vhs function and the putative vhs protein is associated with the virion. Sequence comparison indicated a BHV-1 open reading frame having a 60% similarity to the HSV vhs sequence. This putative BHV-1 open reading frame contained the four conserved regions of the alphaherpesvirus vhs protein. Since an HSV vhs-mutant exhibited less virulence and good immunogenicity, we suggest that a BHV-1 vhs- mutant may hold promising potential as a candidate vaccine.

Reverse immunogenetic and polyepitopic approaches for the induction of cell-mediated immunity against bovine viral pathogens.

The control of several infectious diseases of animals by vaccination is perhaps the most outstanding accomplishment of veterinary medicine in the last century. Even the eradication of some pathogens is in sight, at least in some parts of the world. However, infectious diseases continue to cost millions of dollars to the livestock industry. One of the reasons for the failure to control certain pathogens is the limited emphasis placed on cell-mediated immunity (CMI) in the design of vaccines against these pathogens. Traditionally, vaccine-induced immunity has been studied in relation to antibody-mediated protection. More recent studies, however, have focused on understanding CMI and developing means of inducing CMI. This review focuses on recent advances made in the study of CMI in general and of cytotoxic T lymphocytes in particular. Parallels from studies in human and mouse immunology are drawn in order to point out implications to bovine immunology, specifically for immunity against bovine herpesvirus 1.

An early pseudorabies virus protein down-regulates porcine MHC class I expression by inhibition of transporter associated with antigen processing (TAP).

The objectives of this study were to identify the mechanism(s) of pseudorabies virus (PrV)-induced down-regulation of porcine class I molecules and the viral protein(s) responsible for the effect. The ability of PrV to interfere with the peptide transport activity of TAP was determined by an in vitro transport assay. In this assay, porcine kidney (PK-15) cells were permeabilized with streptolysin-O and incubated with a library of 125I-labeled peptides having consensus motifs for glycosylation in the endoplasmic reticulum (ER). The efficiency of transport of peptides from the cytosol into the ER was determined by adsorbing the ER-glycosylated peptides onto Con A-coupled Sepharose beads. Dose-dependent inhibition of TAP activity was observed in PrV-infected PK-15 cells. This inhibition, which occurred as early as 2 h postinfection (h.p.i.), reached the maximum level by 6 h.p.i., indicating that TAP inhibition is one of the mechanisms by which PrV down-regulates porcine class I molecules. Infection of cells with PrV in the presence of metabolic inhibitors revealed that cycloheximide a protein synthesis inhibitor, but not phosphonoacetic acid a herpesvirus DNA synthesis inhibitor, could restore the cell surface expression of class I molecules, indicating that late proteins are not responsible for the down-regulation. Infection in the presence of cycloheximide followed by actinomycin-D, which results in accumulation of the immediate-early protein, failed to down-regulate class I, indicating that one or more early proteins are responsible for the down-regulation of class I molecules.

The leukotoxin of Pasteurella haemolytica binds to beta(2) integrins on bovine leukocytes.

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.

Bovine lymphocyte antigen-A11--specific peptide motif as a means to identify cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present 8- to 10-mer viral peptides to antiviral cytotoxic T lymphocytes (CTLs). Identification of the allele-specific peptide motifs (ASPMs) of class I molecules enables the prediction of potential CTL epitopes of a virus from its protein sequences. Based on the bovine herpesvirus 1 (BHV-1) protein sequences that conform to the BoLA-A11 ASPM that we identified previously, potential CTL epitopes of BHV-1 were synthesized for use in cytotoxicity assays with CTLs from BHV-1-immunized calves. A peptide binding assay used to select the peptides that are most likely to be CTL epitopes categorized the peptides into groups of high, intermediate, and low binding capacity. Synthetic peptides stimulated lymphocytes from BHV-1-immunized calves to secrete interferon-gamma. Groups of peptides from the major glycoproteins of BHV-1 restimulated CTLs in vitro and sensitized targets for lysis by means of restimulated bulk CTLs.

Identification of murine cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1.

Major histocompatibility complex (MHC) class I molecules present endogenously derived viral peptides to CD8+ cytotoxic T-lymphocytes (CTLs). The objective of this study was to identify the H-2Dd- and H-2Kd-restricted CTL epitopes of bovine herpesvirus 1 (BHV-1), based on the allele-specific peptide motifs (ASPMs) of the above class I molecules. Nine sequences conforming to the H-2Dd and H-2Kd ASPMs were identified on BHV-1 proteins, and the respective peptides were synthesized. Five of these peptides exhibited moderate to strong binding to the Dd molecule. CTLs generated by BALB/c mice immunized with BHV-1 proteins emulsified in a suitable adjuvant effectively lysed peptide-pulsed syngeneic targets, indicating that these epitopes were generated in vivo. Mice immunized with these peptides emulsified in a suitable adjuvant also developed anti-BHV-1 CTLs. These CTLs identified three veritable CTL epitopes among the "potential epitopes" synthesized based on the ASPMs. The elucidation of the CTL epitopes of BHV-1 should aid in the development of efficacious vaccines against this virus.

Bovine herpesvirus-1 infection affects the peptide transport activity in bovine cells.

Infection of cattle with bovine herpesvirus-1 (BHV-1) impairs the cell-mediated immune response (CMI) of the affected host. We investigated the location of interference of BHV-1 with the major histocompatibility complex (MHC) class I antigen presentation pathway by employing an assay that allows assessment of the peptide transport activity of the Transporter associated with Antigen Presentation (TAP) from the cytoplasm into the endoplasmic reticulum (ER). We found a considerable down-regulation of the peptide transport activity in bovine epithelial cells, taking place as early as 2 h after virus infection. This down-regulation was also dose-dependent, and, at high multiplicities of infection (moi), led to an almost complete shutdown of TAP. By inhibiting peptide transport into the ER, the virus impairs loading of MHC class I molecules and their subsequent egress from the ER to the cell surface. This may lead to defective priming of cytotoxic T lymphocytes. Thus, BHV-1 is yet another member of its family Herpesviridae that selectively interferes with the host's antigen presentation machinery to evade the host's immune response in vivo.

Development of a syngeneic bovine fibroblast cell line: implications for the study of bovine cytotoxic T lymphocytes.

The study of T-cell-mediated cytotoxicity in domestic animals, especially in cattle, has been hampered by the lack of proper restimulatory as well as target systems. While the currently available bovine cell lines have not been typed for the major histocompatibility complex (MHC) class I molecules they express, methods to derive lines of cells obtained from animals that are MHC-typed have not been thoroughly explored. In the present study, we describe a method for the development of cell lines from MHC-typed animals. Cells obtained from the skin of a calf typed as bovine lymphocyte antigen-A11/-A13 were transfected with a plasmid containing the whole genome of simian vacuolating virus 40 (SV40). A cell line was derived from the resultant transfectants. This cell line expressed bovine MHC class I molecules on the cell surface, and SV40 large T antigen in the nucleus. The cells were permissive to the replicative cycle of bovine herpesvirus-1 (BHV-1), and the major glycoproteins of BHV-1 were expressed at expected times after infection. The present study should contribute to the study of cytotoxic T lymphocyte response of cattle to BHV-1 and other intracellular pathogens.

The use of bovine MHC class I allele-specific peptide motifs and proteolytic cleavage specificities for the prediction of potential cytotoxic T lymphocyte epitopes of bovine viral diarrhea virus.

Cell mediated immunity (CMI) is crucial for the defense against viruses. Cytotoxic T lymphocytes (CTLs) play a major role in CMI. They recognize endogenous antigenic peptides presented by antigen presenting cells in association with the major histocompatibility complex (MHC) class I molecules. The elucidation of the sequence of CTL epitopes of viruses should help in designing better vaccines. In this study, we have identified candidate epitopes restricted by five bovine MHC class I molecules that are potentially available for presentation to CTLs. The candidate peptide epitopes were identified by using the computer programs available as a part of the Genetics Computer Group package and applying the information on allele-specific peptide motifs and intracellular enzymatic cleavage patterns to the bovine viral diarrhea virus polyprotein. Several candidate peptides were found for each of the bovine lymphocyte antigens (BoLA)-A11, -A20, -HD1, and -HD6 whereas no peptide was found for BoLA-HD7. Based on this finding, the probable contribution of genomic segments of BVDV to the CTL response and strategies for recombinant vaccines are discussed.

Nonlipopolysaccharide component(s) of Lactobacillus acidophilus stimulate(s) the production of interleukin-1 alpha and tumor necrosis factor-alpha by murine macrophages.

Previous studies in our laboratory suggested that Lactobacillus acidophilus strain DDS-1 (LA1) has a suppressive effect on chemically induced tumors in experimental animals. In an effort to understand the possible mechanisms underlying this effect, we investigated the ability of LA1 to induce the production of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha), which have potent cytocidal and cytostatic effects on tumor cells. The mouse macrophage cell line RAW264.7 was incubated with live or heat-killed cells of four strains of L. acidophilus or Bifidobacterium bifidum. Escherichia coli was used as a source of lipopolysaccharide that is known to induce the above cytokines. The amount of the cytokines present in the culture fluid was quantitated by an enzyme-linked immunosorbent assay. LA1 induced the production of higher levels of IL-1 alpha and TNF-alpha than other lactobacilli and bifidobacteria. Stimulation of the production of the cytokines was not due to the lipopolysaccharide (LPS) component, since LPS at concentrations equivalent to, or 100-fold greater than, that of LA1 induced only negligible amounts of IL-1 alpha and TNF-alpha. These results reveal that non-LPS component(s) of LA1 stimulate(s) the production of IL-1 alpha and TNF-alpha by macrophages, indicating that this organism stimulates the production of immunologic factors.

Bovine herpesvirus 1 downregulates the expression of bovine MHC class I molecules.

The mechanism of immunosuppression induced by bovine herpesvirus 1 (BHV-1) was investigated by studying the effects of the virus on the expression of major histocompatibility complex (MHC) class I molecules. After infection with the virus, the expression of class I molecules was detected by flow cytometry and pulse-chase analysis. A selective downregulation of expression of class I molecules was seen in the infected cells, while the class II expression remained unaffected. The reduction in surface expression was evident as early as 8 hours postinfection, reaching significant levels by 12 hours. The downregulation was seen with a multiplicity of infection as low as 0.1. A modified live vaccine strain of BHV-1 also induced the downregulation of class I expression. Analysis of the viral proteins(s) involved in this downregulation with metabolic inhibitors (cycloheximide or phosphonoacetic acid), suggested that the immediate early and/or early proteins of the virus mediate this effect. Pulse-chase analysis revealed that the synthesis of the class I heavy chain, and the assembly/transport of class I molecules were affected by the virus infection. These results suggest that BHV-1 interferes with the molecular mechanisms involved in the synthesis, and assembly/transport of MHC-class I molecules. This interference with the class I antigen processing pathway might help the virus to evade the cytotoxic T-lymphocyte response of the host.