Alloreactive cytotoxic T lymphocytes recognize epitopes determined by both the alpha helices and beta sheets of the class I peptide binding site.

A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha helices and the beta-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the alpha helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (Kb alpha Ld beta) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha helices of Kb can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing Kb alpha Ld beta. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing Kb alpha Ld beta. Thus, alloreactive CTL did not recognize structures determined by the alpha helices alone or by the beta sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with Kb alpha Ld beta. In addition to the Kb alpha helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with Kb alpha Ld beta. Amino acid 24 is located on the beta 2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and Kb alpha Ld beta identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the alpha helices and beta sheets of the ARS. These interactions are best explained by the influence of the alpha helices and beta sheets on the peptide-binding properties of these antigen-presenting molecules.

Subgroup J avian leukosis virus neutralizing antibody escape variants contribute to viral persistence in meat-type chickens.

We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence.

Retroviral delivery of RNA interference against Marek's disease virus in vivo.

The process of RNA interference (RNAi) has been exploited in cultured chicken cells and in chick embryos to assess the effect of specific gene inhibition on phenotypes related to development and disease. We previously demonstrated that avian leukosis virus-based retroviral vectors are capable of delivering effective RNAi against Marek's disease virus (MDV) in cell culture. In this study, similar RNAi vectors are shown to reduce the replication of MDV in live chickens. Retroviral vectors were introduced into d 0 chick embryos, followed by incubation until hatching. Chicks were challenged with 500 pfu of strain 648A MDV at day of hatch, followed by assays for viremia at 14 d postinfection. Birds were monitored for signs of Marek's disease for 8 wk. A stem-loop PCR assay was developed to measure siRNA expression levels in birds. Delivery of RNAi co-targeting the MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduced MDV viremia in vivo, although to lesser extents than were observed in cell culture. Concomitant reductions in disease incidence also were observed, and the extent of this effect depended on the potency of the MDV challenge virus inoculum. Successful modification of phenotypic traits in live birds with retroviral RNAi vectors opens up the possibility that such approaches could be used to alter the expression of candidate genes hypothesized to influence a variety of quantitative traits including disease susceptibility.

Lymphoid organ size varies among inbred lines 6(3) and 7(2) and their thirteen recombinant congenic strains of chickens with the same major histocompatibility complex.

The objective was to evaluate lymphoid organ size in chickens from a series of 13 recombinant congenic strains (RCS) and their highly inbred parental lines (6(3) and 7(2)). The parental line 6(3) was selected for resistance to tumors induced by Marek's disease virus and avian leukosis viruses, whereas line 7(2) was selected for susceptibility to these tumors. Each RCS on the average contains a random one-eighth of genome from the donor line 7(2). Previous studies have shown that lines 6(3) and 7(2) differ in the size of primary lymphoid organs; i.e., the bursa of Fabricius (BF) and the lobes of the thymus (T) are smaller in line 6(3) than line 7(2). In the current study, the relative size of the T, BF, and spleen was first examined in about 15 males from each of 13 RCS and the 2 parental lines at 60 to 69 d of age. The differences of relative BF, T, and spleen size among the RCS and the parental lines 6(3) and 7(2) differed significantly (P < 0.001). Males and females from 4 RCS and the 2 parental lines were evaluated a second time, and differences in the relative sizes in lymphoid organs among the RCS and parental lines were consistent. In 2 RCS, the size of the T and BF was comparatively large as in line 7(2), leading to the conclusion that different allelic forms at 1 or more loci in these RCS regulate the size of both organs. In 2 other RCS, the BF was large compared with the T, suggesting that allelic forms at some loci in these RCS influence the BF independent of the T. The relative lymphoid organ size among the RCS appeared to cosegregate with the concentration of IgG in the plasma measured previously. The evaluation of genomic variability of these lines is underway, and the RCS are available for research on traits that differ between lines 6(3) and 7(2).

Site-directed mutagenesis by overlap extension using the polymerase chain reaction.

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.

Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.

Differential attenuation of the induction by Marek's disease virus of transient paralysis and persistent neurological disease: a model for pathogenesis studies.

Since different biological characteristics of Marek's disease virus (MDV) are attenuated at different passage levels in cell culture, an analysis of attenuation times provides, in theory, a model for establishing the presence or absence of relationships between characteristics, thus providing a basis to link them to genetic changes in the causative virus. We have used this model to better understand the pathogenesis of the central nervous system infection as well as to evaluate the relationship of clinical neurological disease to various other parameters of MDV infection. Inoculation of 15 x7 crossbred chickens with strain 648A of very virulent plus MDV at different passage levels (between 10 and 100) showed that two neurological syndromes (transient paralysis (TP) and persistent neurological disease), were attenuated at different passage levels. While strain 648A lost the ability to induce TP between 30 and 40 passages in chicken embryo fibroblast cultures, an event closely related with all parameters of MDV infection involving viral replication (early cytolytic infection in lymphoid organs and viral replication in the feather follicle epithelium), the ability to induce persistent neurological disease was lost between 80 and 90 passages in chicken embryo fibroblasts, coincident with the loss of neoplastic lesions in peripheral nerves and other visceral organs. These data strongly suggest that transient paralysis and persistent neurological disease are unrelated and differently regulated. Moreover, comparison of brain changes induced by strain 648A at passage level 30 (TP) and at passage level 40 (no TP) also contributed to a better understanding of which brain alterations are associated with the onset of TP. The use of viruses at different passage levels with varying degrees of attenuation is presented as a useful tool for studying pathogenesis of MDV infection.

Cytotoxic T lymphocyte response in chickens immunized with a recombinant fowlpox virus expressing Marek's disease herpesvirus glycoprotein B.

Previously, we demonstrated that cytotoxic T lymphocytes (CTLs) from MHC: B19B19 and MHC: B21B21 chickens inoculated with a non-oncogenic Marek's disease virus (MDV) vaccine strain, SB-1/12 can lyse syngeneic reticuloendotheliosis virus (REV)-transformed cell lines expressing MDV pp38 or gB genes. In this study, we report the characterization of MDV gB-specific CTLs in chickens immunized with recombinant fowlpox virus expressing MDV gB gene (rFPV-gB). Spleen cells from rFPV-gB inoculated chickens (MHC: B19B19), depleted for CD4+, CD8+, TCR gamma delta+, TCR alpha beta 1+ or TCR alpha beta 2+ cells were used as effector cells in chromium release assays. Effector cells depleted of CD8+ or TCR alpha beta 1+, but not CD4+, TCR gamma delta+ or TCR alpha beta 2+ markedly reduced the percentage of specific release (%SR). Compared to the %SR caused by the SB-1/12-sensitized CTLs, the %SR caused by rFPV-gB-sensitized CTLs was low, but statistically significant. This is a first report on the induction of MDV gB-specific CD8+ CTLs in chickens immunized with rFPV-gB vaccine.

Avian leukosis virus subgroup J infection profiles in broiler breeder chickens: association with virus transmission to progeny.

Profiles of infection with avian leukosis virus subgroup J (ALV-J) and factors that predict virus transmission to progeny were studied. Eggs from an infected broiler breeder flock were hatched at the laboratory. The flock was reared in a floor pen, transferred to laying cages at 22 wk, and inseminated to produce fertile eggs. A cohort of 139 chickens was tested at frequent intervals over a 62-wk period for virus, viral antigens, or antibodies in plasma, cloacal swabs, egg albumen, and embryos. Virus was detected in 7% of chicks at hatch but spread rapidly so that virtually all chicks became infected between 2 and 8 wk of age. Mortality due to myeloid leukosis and related tumors was 22%. Over 40% of the chicks developed persistent infections, whereas the remainder experienced transient infections. Five types of infection profiles were recognized. Novel responses included hens that were positive for virus intermittently or started late in life to shed viral antigens into the cloaca. ALV-J was isolated from 6% of 1036 embryos evaluated between 26 and 62 wk. However, over 90% of the virus-positive embryos were produced between 29 and 34 wk of age. Of 80 hens that produced embryos, 21 produced at least one infected embryo and were identified as transmitters. All but one transmitter hen would have been detected by a combination of viremia, cloacal swab, and albumen tests conducted between 18 and 26 wk. However, virus was transmitted to embryos from hens that were not persistently viremic or that rarely shed viral group-specific antigen into the albumen of their eggs. Intermittent patterns of both antigen shedding and virus transmission to embryos were observed in some hens. These results validate current screening procedures to identify potential transmitter hens and provide some suggestions for improvement but also show that identification of all transmitter hens by such procedures is unlikely. Thus, eradication programs based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks.

Poultry vaccines of the future.

Current poultry vaccines are based either on live attenuated organisms or on killed organisms. Future vaccines also may be based on deletion mutants, live viral or bacterial vectors that express foreign genes, and naked DNA. Vaccines have different purposes, depending on the disease, which govern their intrinsic characteristics. Improvement of vaccine efficacy can be addressed by modifications of the vaccine and its administration, modifications in the capacity of the host to mount an immune response, and modifications of environmental factors. The concept of "designer vaccines" for matching vaccines that deliver specific antigenic peptides to chickens with the MHC haplotype that best presents those peptides to T cells is discussed.

Chicken major histocompatibility complex class I definition using antisera induced by cloned class I sequences.

Alloantisera directed against chicken class I MHC (BFIV) antigens were produced by using transfected cell lines expressing cloned BFIV sequences. The cloned BFIV sequences were from haplotypes *12, *13, and *21. Two laboratory-derived class I mutant sequences (BFIV13m126 and BFIV21m78) were developed to analyze cross-reactive epitopes and to induce specific alloantisera. Antisera were tested in hemagglutination and flow cytometry assays. The antisera produced were highly specific and had minimal cross-reactivity. The antisera induced by the BF1V21m78 mutant confirmed the significance of amino acids 78 and 81 in cross-reactivity between haplotypes B*21 and B*5. The highly specific antisera were tested by hemagglutination on red blood cells of 31 different MHC haplotypes. The consistency of hemagglutination patterns and minimal cross-reactivity demonstrated that these BFIV antisera are extremely valuable in defining MHC haplotype in various chicken lines. Because of the extreme low level of recombination between the chicken class I and class II loci, identification of BFIV allele can be used to define MHC haplotype within a line. Complete identity between the transfected cell line and the chicken used to produce the antiserum is required to ensure the monospecificity.

A review of the development of chicken lines to resolve genes determining resistance to diseases.

The resolution of genes that determine resistance to disease is described using chicken lines maintained at the Avian Disease and Oncology Laboratory (ADOL). This description includes a summary 1) of existing selected and inbred lines differing for resistance to viral-induced tumors, i.e., Marek's disease (MD) and lymphoid leukosis (LL), and of the use of inbred and line crosses to define relevant disease-resistant genes, e.g., TV, ALVE, B, R, LY4, TH1, BU1, and IGG1; 2) of the development of TVB*/ALVE congenic lines to establish the affects of endogenous virus (EV) expression on resistance to avian leukosis virus (ALV), and methods to detect ALVE expression; 3) of the development of B congenic lines to define the influence of the MHC on MD resistance and vaccinal immunity, for producing B antisera, and for evaluating DNA sequences of Class I and II genes; and 4) of the current development of 6C.7 recombinant congenic strains (RCS) to define the role of non-MHC genes influencing susceptibility to MD and LL tumors, immune competence, and epistatic effects of genes. The procedures of pedigree mating, to avoid or maintain inbreeding, and of blood-typing, to ensure genetic purity of the lines, are also described.

Identification of class II major histocompatibility complex polymorphisms predicted to be important in peptide antigen presentation.

Chickens of the B2, B5, B15, B19, and B21 B-congenic haplotypes differ in disease resistance. Complementary DNA from B-congenic chicken strains have been analyzed for allelic diversity of expressed Class II MHC genes. The predicted amino acid sequences of eight genes from five haplotypes were subjected to Wu-Kabat variability analysis. The B-L gene polymorphic regions and conserved regions are highly similar to the human leukocyte antigen Class II genes. Therefore, the present analysis reveals candidate polymorphisms important in determining the spectrum of antigenic peptides presented to T helper cells, and allelic differences possibly important in resistance to avian disease.

Development and validation of a PCR-RFLP assay to evaluate TVB haplotypes coding receptors for subgroup B and subgroup E avian leukosis viruses in White Leghorns.

The cellular receptor of subgroup B avian leukosis virus (ALVB) is encoded by a gene at the tumour virus B (TVB) locus. TVB alleles encode specific receptors permitting infection by exogenous ALVB or avian leukosis virus subgroup D (ALVD) as well as endogenous avian leukosis virus subgroup E (ALVE), and thus susceptibility is dominant to resistance. Two single nucleotide polymorphisms at the TVB locus have been reported distinguishing three TVB alleles (TVB*S1, TVB*S3 and TVB*R). We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using the two single nucleotide polymorphisms to define three observed allelic haplotypes and to identify the six possible TVB genotypes consisting of the three haplotypes in defined laboratory strains of chickens. One additional potential allelic haplotype and four genotypes were also briefly discussed. Chickens from parents heterozygous for different TVB alleles were challenged with Rous sarcoma viruses of subgroup ALVB and ALVE to induce wing-web tumours. Tumour incidences were evaluated between chickens of the genotypes determined with this newly developed PCR-RFLP assay. Importantly, chickens typed with this assay as TVB*S3/*S3 were resistant to infection by ALVE only, and those TVB*R/*R were resistant to both ALVE and ALVB. Furthermore, a vast majority of chickens with the susceptible TVB*S1/- genotypes developed a tumour. This PCR-RFLP assay enables a relatively rapid assessment of all six anticipated TVB genotypes in experimental strains of chickens undergoing segregation for TVB*S1, TVB*S3, and TVB*R alleles. This non-infectious assay should be further evaluated for the capacity to select and breed commercial chickens for genetic resistance to infections by ALVB, ALVD and ALVE.