Two doses of bovine viral diarrhea virus DNA vaccine delivered by electroporation induce long-term protective immune responses.

Bovine viral diarrhea virus (BVDV) is a pathogen of major importance in cattle, so there is a need for new effective vaccines. DNA vaccines induce balanced immune responses and are relatively inexpensive and thus promising for both human and veterinary applications. In this study, newborn calves with maternal antibodies were vaccinated intramuscularly (i.m.) with a BVDV E2 DNA vaccine with the TriGrid Delivery System for i.m. delivery (TDS-IM). Two doses of this vaccine spaced 6 or 12 weeks apart were sufficient to induce significant virus-neutralizing antibody titers, numbers of activated T cells, and reduction in viral shedding and clinical presentations after BVDV-2 challenge. In contrast to the placebo-treated animals, the vaccinated calves did not lose any weight, which is an excellent indicator of the well-being of an animal and has a significant economic impact. Furthermore, the interval between the two vaccinations did not influence the magnitude of the immune responses or degree of clinical protection, and a third immunization was not necessary or beneficial. Since electroporation may enhance not only the magnitude but also the duration of immunity after DNA immunization, the interval between vaccination and challenge was extended in a second trial, which showed that two doses of this E2 DNA vaccine again significantly reduced clinical disease against BVDV for several months. These results are promising and support this technology for use against infectious diseases in cattle and large species, including humans, in general.

Respiratory disease caused by Mycoplasma bovis is enhanced by exposure to bovine herpes virus 1 (BHV-1) but not to bovine viral diarrhea virus (BVDV) type 2.

To determine if previous exposure to bovine viral diarrhea virus (BVDV) and bovine herpes virus 1 (BHV-1) type 2 affects the onset of disease caused by Mycoplasma bovis, 6- to 8-month-old beef calves were exposed to BVDV or BHV-1 4 d prior to challenge with a suspension of 3 clinical isolates of M. bovis. Animals were observed for clinical signs of disease and at necropsy, percent abnormal lung tissue and presence of M. bovis were determined. Most animals pre-exposed to BHV-1 type 2 but not BVDV developed M. bovis-related respiratory illness. In a second trial, we determined that a 100-fold reduction in the number of M. bovis bacteria administered to BHV-1 exposed animals reduced the percentage of abnormal lung tissue but not the severity of clinical signs. We conclude that previous exposure to BHV-1 but not BVDV type 2 was a necessary cause of M. bovis-related respiratory diseases in our disease model.

Electroporation enhances immune responses and protection induced by a bovine viral diarrhea virus DNA vaccine in newborn calves with maternal antibodies.

Bovine viral diarrhea virus (BVDV) is one of the major pathogens in cattle. In this study, newborn calves with maternal antibodies were vaccinated with a BVDV DNA vaccine, either by conventional intramuscular (IM) injection or with the TriGrid™ Delivery System for IM delivery (TDS-IM). The calves vaccinated with the TDS-IM developed more rapidly and effectively BVDV-specific humoral and cell-mediated immune responses in the presence of maternal antibodies. Overall, the immune responses induced by delivery with the TDS-IM remained stronger than those elicited by conventional IM injection of the BVDV DNA vaccine. Accordingly, electroporation-mediated delivery of the BVDV DNA vaccine resulted in close to complete protection from clinical signs of disease, while conventional IM administration did not fully prevent morbidity and mortality following challenge with BVDV-2. These results demonstrate the TDS-IM to be effective as a delivery system for a BVDV DNA vaccine in newborn calves in the presence of maternal antibodies, which supports the potential of electroporation as a delivery method for prophylactic DNA vaccines.

A UL47 gene deletion mutant of bovine herpesvirus type 1 exhibits impaired growth in cell culture and lack of virulence in cattle.

Tegument protein VP8 encoded by the U(L)47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U(L)47 gene-deleted BHV-1 in cultured cells and its natural host. The U(L)47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U(L)47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U(L)47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U(L)47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U(L)47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U(L)47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.

Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22.

The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

Requirement of N-myristoyltransferase 1 in the development of monocytic lineage.

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.

Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29.

Protein myristoylation is a co-translational process, catalyzed by N-myristoyltransferase (NMT) that occurs after the initiating methionine is removed by methionine aminopeptidase (MetAP). The enzymes NMT and MetAP play a major role in the process of myristoylation of oncoproteins including the c-src family. In this study, we examined the levels of expression of MetAP2, NMT, and NMT inhibitor protein 71 (NIP71) in human colon cancer cell lines (HCCLs). We examined the influence of cell density on the expression of the above proteins in HT29 cells. Western blot analysis of MetAP2 and NMT demonstrated higher levels of protein expression in low density of HT29 while low expression in high density was observed. In addition, we observed that NIP71 and pp60(c-src) expressions were dependent on the cell density of HT29. This is the first study demonstrating the expression of MetAP2, NMT, pp60(c-src), and NIP71 in HCCLs.