A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus.

Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.

Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection.

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection.

Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human.

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

In vitro and in vivo effects of recombinant bovine interferon-tau on bovine leukemia virus.

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.

Cell dynamics and immune response to BLV infection: a unifying model.

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Increase in gammadelta T cells in the blood of cattle persistently infected with bovine leukemia virus following administration of recombinant bovine IFN-gamma.

To examine the effect of recombinant bovine interferon-gamma (rbIFN-gamma) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-gamma. All cattle were febrile after inoculation with IFN-gamma and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased for 2-3 days and then their numbers were recovered. The number of gammadelta T cells increased after the fever. In contrast, the number of IgM+ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-gamma administration. These results suggest that IFN-gamma induces the up-regulation of gammadelta T cells, decreases the number of IgM+ lymphocytes and suppresses the growth of BLV in BLV-infected cattle in vivo.