The Mut UL5-I682R Marek's Disease Virus with a Single Nucleotide Mutation Within the Helicase-Primase Subunit Gene not only Reduces Virulence but also Provides Partial Vaccinal Protection Against Marek's Disease.

Marek's disease virus (MDV) is an oncogenic herpesvirus that afflicts chickens with the disease known as Marek's disease (MD). This virus induces tumors, nerve lesions, immunosuppression, and death of affected birds. Vaccines are the primary control method for MD but, due to the periodic evolution of field strains, it is necessary to explore the development of new MD vaccines. MD vaccines are often attenuated MDV strains generated through serial passage in vitro. We previously used experimental evolution of MDV to provide a better understanding of the genetic basis of attenuation. During complete genome sequencing of evolved MDV populations, we identified a point mutation within the UL5 helicase-primase gene and created a UL5 recombinant virus that significantly reduced disease incidence by 89%-100%. To determine if experimental evolution also identifies mutations that provide protective qualities as potential vaccine candidates, we tested the UL5 recombinant virus as a vaccine and compared its protection to commercial herpesvirus of turkey (HVT) and bivalent (HVT + SB-1) vaccines. Both commercial vaccines resulted in higher protection against MD than did the UL5 recombinant virus, although the UL5 virus did provide protection against developing MD in 46%-70% of birds challenged. This indicates that a mutation within the UL5 helicase-primase gene not only reduces virulence but also confers protection against challenge with virulent MDV, providing support that not only can experimental evolution identify candidate mutations involved in attenuation but can also identify potential candidates for use in vaccine development.

Addition of a UL5 helicase-primase subunit point mutation eliminates bursal-thymic atrophy of Marek's disease virus ∆Meq recombinant virus but reduces vaccinal protection.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus and the causative agent of Marek's disease (MD), characterized by immunosuppression, paralysis, nerve enlargement and induction of T-cell lymphomas in chickens. Despite widespread usage of vaccines since the 1970s to control MD, more virulent field strains of MDV have emerged that overcome vaccinal protection, necessitating the development of new and more protective MD vaccines. The ∆Meq virus, a recombinant Md5 strain MDV lacking the viral oncogene Meq, is one candidate MD vaccine with great potential but unfortunately it also causes bursal-thymic atrophy (BTA) in maternal antibody negative chickens, raising concerns that impede commercial use as a vaccine. Previously, we identified a point mutation within UL5 that reduced in vivo replication in attenuated viruses. We proposed that introduction of the UL5 point mutation into the ∆Meq virus would reduce in vivo replication and eliminate BTA yet potentially retain high protective abilities. In birds, the ∆Meq+UL5 recombinant MDV had reduced replication compared to the original ∆Meq virus, while weights of lymphoid organs indicated that ∆Meq+UL5 did not induce BTA, supporting the hypothesis that reduction of in vivo replication would also abolish BTA. Vaccine trials of the ∆Meq+UL5 virus compared to other ∆Meq-based viruses and commercial vaccines show that, while the ∆Meq+UL5 does provide vaccinal protection, this protection was also reduced compared to the original ∆Meq virus. Therefore, it appears that a very delicate balance is required between levels of replication able to induce high vaccinal protection, yet not so high as to induce BTA.

Chlamydia and antigenic mimicry.

Chlamydial infections are among the most common human infections. Every year, in millions of humans, they cause infections of the eyes, the respiratory tract, the genital tract, joints, and the vasculature. Chlamydiae are obligate intracellular prokaryotic pathogens. Chlamydiae promote, in susceptible host cells that include mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, and monocytes and macrophages, their survival while causing disease of varying clinical importance and consequence in their hosts. Chlamydia infections often precede the initiation of autoimmune diseases, and Chlamydiae are often found within autoimmune lesions. Thus, they have been suspected in the etiology and pathogenesis of autoimmune diseases. Autoimmune diseases have many causes. Genes, notably genes encoding cell-surface proteins that display peptides for immune recognition, the major histocompatibility complex (MHC), the environment, and the microbial diversity within the human body determine the susceptibility to autoimmune diseases. One mechanism by which infection is linked to the initiation of autoimmunity is termed molecular mimicry. Molecular mimicry describes the phenomenon of protein products from dissimilar genes sharing similar structures that elicit an immune response to both self and microbial proteins. Molecular mimicry might thus be a mechanism by which infections trigger autoimmune diseases. For the purpose of this chapter, we will focus on chlamydial proteins that mimic host self-proteins and thus contribute to initiation and maintenance of autoimmune diseases. Thus far, the strongest cases for molecular mimicry seem to have been made for chlamydial heat shock proteins 60, the DNA primase of Chlamydia trachomatis, and chlamydial OmcB proteins.

Sustained CD8+ T-cell immune response to a novel immunodominant HLA-B*0702-associated epitope derived from an Epstein-Barr virus helicase-primase-associated protein.

Generation of a peptide-based vaccine against persistent viral infections, such as Epstein-Barr virus (EBV), requires identification of immunodominant epitopes recognized by anti-viral cytotoxic T-cells. Using available computer algorithms, we have screened the entire translated EBV genome for potential HLA-B7-binding peptides. The binding to HLA-B7 of 18 selected peptides was assessed by competitive binding assays and was found to correlate with the computer-assigned scores, confirming the predictive value of these algorithms in selection of HLA-B7-associated peptides. Screening of the immune responses to these peptides by ELISpot assays identified a novel immunodominant epitope, termed LPRA, derived from an EBV helicase-primase-associated protein encoded by BBLF2/3. Peptide-specific cells constituted up to 0.8% LPRA-specific CD8+ T-cells in the matured anti-viral response. Cytotoxic and proliferative cytotoxic T lymphocytes (CTL) responses to the LPRA peptide were readily demonstrated ex vivo. In addition, mutational studies of this epitope demonstrated a highly specific recognition by LPRA-specific CD8+ T-cells. Taken together, our data suggest that the novel lytic-phase HLA-B7-associated epitope contains essential features required of a component in an EBV peptide-based vaccine.

Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication.

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.