Ten-Gram-Scale Total Synthesis of the Anticancer Drug Candidate E7130 to Supply Clinical Trials.

E7130 is a novel drug candidate with an exceedingly complex chemical structure of the halichondrin class, discovered by a total synthesis approach through joint research between the Kishi group at Harvard University and Eisai. Only 18 months after completion of the initial milligram-scale synthesis, ten-gram-scale synthesis of E7130 was achieved, providing the first good manufacturing practice (GMP) batch to supply clinical trials. This paper highlights the challenges in developing ten-gram-scale synthesis from the milligram-scale synthesis.

Influence of BoLA-DRB3 Polymorphism and Bovine Leukemia Virus (BLV) Infection on Dairy Cattle Productivity.

Enzootic bovine leukosis caused by the bovine leukemia virus (BLV) results in substantial damage to the livestock industry; however, we lack an effective cure or vaccine. BoLA-DRB3 polymorphism in BLV-infected cattle is associated with the proviral load (PVL), infectivity in the blood, development of lymphoma, and in utero infection of calves. Additionally, it is related to the PVL, infectivity, and anti-BLV antibody levels in milk. However, the effects of the BoLA-DRB3 allele and BLV infection on dairy cattle productivity remain poorly understood. Therefore, we investigated the effect of BLV infection and BoLA-DRB3 allele polymorphism on dairy cattle productivity in 147 Holstein dams raised on Japanese dairy farms. Our findings suggested that BLV infection significantly increased milk yield. Furthermore, the BoLA-DRB3 allele alone, and the combined effect of BLV infection and the BoLA-DRB3 allele had no effect. These results indicate that on-farm breeding and selection of resistant cattle, or the preferential elimination of susceptible cattle, does not affect dairy cattle productivity. Additionally, BLV infection is more likely to affect dairy cattle productivity than BoLA-DRB3 polymorphism.

Anti-BLV antibodies in whey correlate with bovine leukemia virus disease progression and BoLA-DRB3 polymorphism.

Introduction: Bovine leukemia virus (BLV) belongs to the family Retroviridae and is a causative agent for enzootic bovine leucosis, the most common neoplastic disease affecting cattle worldwide. BLV proviral load (PVL) is associated with disease progression and transmission risk but requires blood collection and quantitative PCR testing. Anti-BLV antibodies in whey have been used as a diagnostic tool for BLV infection; however, quantitative utilization has not been fully investigated. Furthermore, bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and PVL, but its effect on anti-BLV antibody levels in whey from BLV infected dams is unknown. Therefore, we aimed to investigate whether it is possible to correctly predict PVL in the blood and milk based on the amount of anti-BLV antibodies in milk, and whether the BoLA-DRB3 alleles associate with the amount of anti-BLV antibodies in milk. Methods: We examined whey from 442 dams from 11 different dairy farms located in 6 prefectures in Japan, including susceptible dams carrying at least one BoLA-DRB3* 012:01 or * 015:01 allele related with high PVL, resistant dams carrying at least one BoLA-DRB3 * 002:01, * 009:02, or * 014:01:01 allele related with low PVL, and neutral dams carrying other alleles. Results: First, our results provided compelling evidence that anti-BLV antibody levels in whey were positively correlated with the anti-BLV antibody levels in serum and with BLV PVL in blood and milk, indicating the possibility of estimating BLV PVL in blood and milk by measuring anti-BLV antibody levels in whey. Thus, our results showed that antibody titers in milk might be effective for estimating BLV transmission risk and disease progression in the field. Second, we demonstrated that anti-BLV antibody levels in whey from BLV resistant dams were significantly lower than those from susceptible and neutral dams. Discussion: This is the first report suggesting that the BoLA-DRB3 polymorphism affects anti-BLV antibody levels in whey from BLV-infected dams. Taken together, our results suggested that anti-BLV antibody levels in whey, measured by enzyme-linked immunosorbent assay, may be a useful marker to diagnose the risk of BLV infection and estimate PVL in blood and milk.

No evidence of bovine leukemia virus proviral DNA and antibodies in human specimens from Japan.

BACKGROUND: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential. RESULT: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples. CONCLUSION: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV.

BoLA-DRB3 Polymorphism Controls Proviral Load and Infectivity of Bovine Leukemia Virus (BLV) in Milk.

Bovine leukemia virus (BLV), which causes enzootic bovine leukosis, is transmitted to calves through the milk of BLV-infected dams. Bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and proviral load (PVL). However, the effect of BoLA-DRB3 polymorphism on the infectivity and PVL of milk from BLV-infected dams remains unknown. This study examined milk from 259 BLV-infected dams, including susceptible dams carrying at least one BoLA-DRB3*012:01 or *015:01 allele with high PVL, resistant dams carrying at least one BoLA-DRB3*002:01, *009:02, or *014:01:01 allele with low PVL, and neutral dams carrying other alleles. The detection rate of BLV provirus and PVL were significantly higher in milk from susceptible dams than in that from resistant dams. This result was confirmed in a three-year follow-up study in which milk from susceptible dams showed a higher BLV provirus detection rate over a longer period than that from resistant dams. The visualization of infectivity of milk cells using a luminescence syncytium induction assay showed that the infectious risk of milk from BLV-infected dams was markedly high for susceptible dams compared to resistant ones. This is the first report confirming that BoLA-DRB3 polymorphism affects the PVL and infectivity of milk from BLV-infected dams.

Association of Bovine Leukemia Virus-Induced Lymphoma with BoLA-DRB3 Polymorphisms at DNA, Amino Acid, and Binding Pocket Property Levels.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis, a malignant B-cell lymphoma in cattle. The DNA sequence polymorphisms of bovine leukocyte antigen (BoLA)-DRB3 have exhibited a correlation with BLV-induced lymphoma in Holstein cows. However, the association may vary between different cattle breeds. Furthermore, little is known about the relationship between BLV-induced lymphoma and DRB3 at the amino acid and structural diversity levels. Here, we comprehensively analyzed the correlation between BLV-induced lymphoma and DRB3 at DNA, amino acid, and binding pocket property levels, using 106 BLV-infected asymptomatic and 227 BLV-induced lymphoma Japanese black cattle samples. DRB3*011:01 was identified as a resistance allele, whereas DRB3*005:02 and DRB3*016:01 were susceptibility alleles. Amino acid association studies showed that positions 9, 11, 13, 26, 30, 47, 57, 70, 71, 74, 78, and 86 were associated with lymphoma susceptibility. Structure and electrostatic charge modeling further indicated that binding pocket 9 of resistance DRB3 was positively charged. In contrast, alleles susceptible to lymphoma were neutrally charged. Altogether, this is the first association study of BoLA-DRB3 polymorphisms with BLV-induced lymphoma in Japanese black cattle. In addition, our results further contribute to understanding the mechanisms regarding how BoLA-DRB3 polymorphisms mediate susceptibility to BLV-induced lymphoma.

Bovine major histocompatibility complex (BoLA) heterozygote advantage against the outcome of bovine leukemia virus infection.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis. Host genetic heterozygosity at the major histocompatibility complex can enhance the ability to combat infectious diseases. However, heterozygote advantage is loci specific and depends on disease type. Bovine leukocyte antigen (BoLA)-DRB3 polymorphisms are related with BLV-infection outcome; however, whether BoLA-DRB3 heterozygotes have an advantage against BLV-induced lymphoma and proviral load (PVL) remains unclear. By analyzing 1567 BLV-infected individuals, we found that BoLA-DRB3 heterozygous status was significantly associated with lymphoma resistance irrespective of cattle breeds (p < 0.0001). Similarly, decreased PVL was observed in BoLA-DRB3 heterozygotes (p = 0.0407 for Holstein cows; p = 0.0889 for Japanese Black cattle). Our report provides first evidence of BoLA-DRB3 heterozygote advantage against BLV infection outcome.

Detection and molecular characterization of bovine leukemia virus in beef cattle presented for slaughter in Egypt.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most common neoplastic disease of cattle worldwide and a serious problem for the cattle industry. Previous studies have shown the molecular prevalence of BLV and the coexistence of BLV genotype-1 and -4 in Egyptian dairy cattle; however, the molecular characteristics of BLV in Egyptian beef cattle are unknown. Therefore, we collected blood samples of 168 beef cattle from slaughterhouses in three governorates in Egypt. Based on BLV-CoCoMo-qPCR-2 targeting long terminal repeats and nested PCR targeting the env-gp51 gene, the BLV provirus infection rates were found to be 47/168 (28.0%) and 42/168 (25.0%), respectively. Phylogenetic analysis based on 501 bp of the BLV env-gp51 gene from 42 BLV isolates revealed that at least six distinctive strains (b, e, f, g, x, and z) were prevalent in cattle across the examined regions. Furthermore, phylogenetic analysis of the 420 bp sequence of the BLV env-gp51 region of the six strains against 11 known genotypes showed that the strains b, e, f, and g were clustered into genotype-1, and strains x and z were clustered into genotype-4. Our results also indicated that strains b and x exist in both dairy and beef cattle in Egypt. The present study is the first to detect and genotype BLV among beef cattle in Egypt.

Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams.

Bovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission.

A landmark in drug discovery based on complex natural product synthesis.

Despite their outstanding antitumour activity in mice, the limited supply from the natural sources has prevented drug discovery/development based on intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) on a >10 g scale with >99.8% purity under GMP conditions. Interestingly, E7130 not only is a novel microtubule dynamics inhibitor but can also increase intratumoural CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. According to these unique effects, E7130 significantly augment the effect of antitumour treatments in mouse models and is currently in a clinical trial. Overall, our work demonstrates that a total synthesis can address the issue of limited material supply in drug discovery/development even for the cases of complex natural products.

A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus.

BACKGROUND: Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). METHODS: We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. RESULTS: We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. CONCLUSION: The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

Oral administration of immunopotentiator from Pantoea agglomerans 1 (IP-PA1) improves the survival of B16 melanoma-inoculated model mice.

To investigate the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1) as a supportive drug in melanoma therapy, we analyzed the immunological effects of IP-PA1 on melanoma-inoculated model mice. Oral administration of IP-PA1 increased the serum levels of tumor necrosis factor (TNF)-α at 2 h after the administration and interferon (IFN)-γ and IL-12 at 12 h after the administration in naïve BALB/cCrSlc mice as evaluated by ELISA. IP-PA1 did not affect the proliferation of melanoma cells directly determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Combinatory treatment of IP-PA1 with doxorubicin for 9 days increased the serum levels of IFN-γ and IL-12 by 71.0 and 15.3%, respectively, compared to the treatment of doxorubicin alone in melanoma-bearing C57BL/6NCrSlc mice as evaluated by ELISA. It also increased the proportion of natural killer (NK) cells and the ratio of CD4(+) to CD8(+) T cells in the spleen from 6.1 ± 0.3 to 7.4 ± 0.5% and from 1.25 ± 0.03 to 1.38 ± 0.04, respectively, compared to the treatment of doxorubicin alone as analyzed by flow cytometry. The mean survival period of melanoma-bearing, doxorubicin treated mice was prolonged from 31.4 ± 7.1 to 35.3 ± 8.4, 51.1 ± 5.4, and 45.0 ± 8.4 days by combinatory treatment of IP-PA1 at the daily doses of 0.1, 0.5, and 1 mg/kg, respectively. In conclusion, the results of the present study suggest the usefulness of IP-PA1 as a supportive drug in melanoma therapy.

Recovery from immunosuppression-related disorders in humans and animals by IP-PA1, an edible lipopolysaccharide.

Immunopotentiator from Pantoea agglomerans 1 (IP-PA1), an edible lipopolysaccharide (LPS) derived from symbiotic bacteria in crops, is a promising immunomodulator. It activates macrophages and protects from chemotherapeutic agent-induced growth inhibition in macrophages in vitro. We showed the immune-recovery effects of IP-PA1 in a chicken model of dexamethasone-induced stress in which IP-PA1 inhibited thymic and bursal atrophy and improved antibody production in response to vaccination. Furthermore, we showed IP-PA1 improved survival of melanoma-bearing, doxorubicin-treated mice, although not directly affecting the proliferation of melanoma cells, dominantly through the improvement of host antitumor immunity. These results suggest that IP-PA1 could have other possible applications in the treatment of various immunosuppression-related disorders in humans and animals.

Protective effects of the immunopotentiator from Pantoea agglomerans 1 on chemotherapeutic agent-induced macrophage growth inhibition.

BACKGROUND: The immunopotentiator from Pantoea agglomerans 1 (IP-PA1) is an edible lipopolysaccharide (LPS) derived from symbiotic bacteria found in crops. IP-PA1 is known to ameliorate chemotherapy-induced immunosuppression; therefore, its macrophage-activating effect in the presence of chemotherapeutic agents was evaluated. MATERIALS AND METHODS: Nuclear factor-kappaB (NF-kappaB) activation in IP-PA1-treated RAW264 and J774.1 cells was examined using Western blot analyses; Griess assay and ELISA were used to examine the production of nitric oxide and tumour necrosis factor alpha, respectively. The expression of apoptosis-related proteins was also assessed using Western blot analyses. The effect of IP-PA1 on doxorubicin-induced apoptosis was analyzed by flow cytometry after annexin-V staining. The growth of macrophages treated with chemotherapeutic agents and IP-PA1 was analyzed using an MTT assay. RESULTS: IP-PA1 activated NF-kappaB and ameliorated chemotherapy induced growth inhibition in the cells. CONCLUSION: IP-PA1 is an edible drug that can potentially support chemotherapy by ameliorating chemotherapy-induced immunosuppression.

Pretreatment of macrophages with the combination of IFN-gamma and IL-12 induces resistance to Leishmania major at the early phase of infection.

Interferon (IFN)-gamma is essential but not sufficient to control leishmaniasis. It is known that IFN-gamma is one of the major macrophage-activating cytokines, and the activated macrophages are a principal source of interleukin (IL)-12, which induces autocrine macrophage activation. In this study, the combined effect of IFN-gamma and IL-12 on the susceptibility of macrophages to Leishmania major infection was evaluated. Macrophages pretreated with IFN-gamma and/or IL-12 were infected with the parasites. Four hr post-infection (p.i.), the levels of infection and parasite load in the macrophages treated with the combination of IFN-gamma and IL-12 (IFN-gamma/IL-12) were significantly lower than those in the nontreated cells. However, the macrophages treated with either IFN-gamma or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-gamma/IL-12-treated and IFN-gamma-treated macrophages showed significantly lower levels of infection and parasite load than the nontreated cells, and higher levels of resistance was observed in the IFN-gamma/IL-12-treated macrophages than in the IFN-gamma-treated macrophages. Although IFN-gamma/IL-12 treatment of macrophages prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to the macrophage culture. These results suggest that IFN-gamma/IL-12 treatment prior to the infection restricts the early phase of the infection.

Cell surface expression of a chimeric protein containing mouse immunoglobulin G1 Fc domain and its immunological property.

Recently we reported that a chimeric molecule containing mouse transferrin receptor and immunoglobulin G1 (IgG1) Fc, mTR-Fc, induced higher immune responses and can be used as a vaccine adjuvant. In this study, the immunological property of the molecule was investigated. Although, the mTR-Fc did not activate complement classical pathway, it was recognized by activated macrophage as like intact IgG Fc, which is recognized by macrophage via Fcgamma receptor. In addition, we found that splenocyte simultaneously exposed to lipopolysaccaride (LPS) and mTR-Fc produced higher amount of interleukin-10, comparing to that exposed to only LPS. These results suggest that the mTR-Fc molecules conserved the IgG Fc property to biasing immune responses via modulation of cytokine production by antigen presenting cell.

Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection.

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.

Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes.

Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice.

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.

Induction of apoptosis in rabbit kidney cell under high-level expression of bovine herpesvirus 1 U(s) ORF8 product.

OBJECTIVE: The open reading frame 8 (ORF8) within the short unique (U(s)) region of the bovine herpesvirus 1 (BHV-1) genome was predicted to encode a protein with homology to U(s)9 protein of other alpha herpesviruses. The aim of this study is to identify the protein encoded by the U(s) ORF8 and to examine the effect of its expression in mammalian cells. METHODS: A polyclonal antiserum was raised against U(s) ORF8 protein expressed in Escherichia coli. A recombinant baculoviurs designated as Ac/CA8 was created by integrating U(s) ORF8 under the control of the mammalian CAG promoter into the baculovirus genome. U(s) ORF8 protein was expressed in rabbit kidney (RK13) cells transduced by Ac/CA8. RESULTS: The antiserum reacted specifically with 27-and 32-kD polypeptides from the BHV-1-infected and Ac/CA8-transduced RK13 cells. High-level expression of U(s) ORF8 protein in RK13 cells transduced by Ac/CA8 led to a distinct cytopathic effect and a reduction in cell viability; the onset of apoptotic cell death was induced as judged by DNA laddering and chromatin condensation analyses. CONCLUSIONS: These data represent the identification of BHV-1 U(s )ORF8 protein, which directly induces apoptosis in RK13 cells.