Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70.

We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.     

The structure of cloned 3'-terminal RNA region of bovine leukemia virus (BLV)

cDNA synthesized on the bovine leukemia virus RNA template has been cloned in the pBR322 Pst I site. Colony hybridization with BLV RNA fragments and oligo (dT) has revealed a clone with cDNA insert containing 660 3'-terminal nucleotides of the BLV genome. The nucleotide sequence of the insert corresponding to U3 and R regions of the long terminal repeats (LTR) of viral genome has been determined. BLV U3, like U3 of other retroviruses, presumably contains promoter. The unusually long R region (about 230 bp), a certain homology with ATLV U3-R and some other structural features allow to group BLV LTR together with ATLV LTR in a separate class of retroviral LTR.     

Augmentation of bovine leukemia virus (BLV)-specific lymphocyte proliferation responses in ruminants by inoculation with BLV env-recombinant vaccinia virus: their role in the suppression of BLV replication.

Lymphocyte proliferation responses were investigated in sheep and cattle, in which the replication of bovine leukemia virus (BLV) had been known to be suppressed by inoculation with recombinant vaccinia virus (rVV) expressing BLV envelope glycoprotein (gp60). Enhanced lymphocyte proliferation responses were observed in animals inoculated with rVV, regardless of whether they were naive or BLV carriers. These responses were roughly inversely correlated to the growth of BLV in the peripheral blood leukocytes. In contrast, there was no apparent correlation between humoral immune response and BLV growth. Based on these results, it was suggested that rVV rendered its suppressive effect of BLV replication primarily via augmentation of cell-mediated immunity.     

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).     

Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL.

Two ALVAC (canarypox virus)-based recombinant viruses expressing the feline leukemia virus (FeLV) subgroup A env and gag genes were assessed for their protective efficacy in cats. Both recombinant viruses contained the entire gag gene. ALVAC-FL also expressed the entire envelope glycoprotein, while ALVAC-FL(dl IS) expressed an env-specific gene product deleted of the putative immunosuppressive region. Although only 50% of the cats vaccinated with ALVAC-FL(dl IS) were protected against persistent viremia after oronasal exposure to a homologous FeLV isolate, all cats administered ALVAC-FL resisted the challenge exposure. Significantly, protection was afforded in the absence of detectable FeLV-neutralizing antibodies. These results represent the first effective vaccination of cats against FeLV with a poxvirus-based recombinant vector and have implications that are relevant not only to FeLV vaccine development but also to developing vaccines against other retroviruses, including human immunodeficiency virus.     

Comparison of bovine leukemia virus (BLV) and CMV promoter-driven reporter gene expression in BLV-infected and non-infected cells

Background  

Viral promoters are used in mammalian expression vectors because they generally have strong activity in a wide variety of cells of differing tissues and species.     

Using PCR for early diagnosis of bovine leukemia virus infection in some native cattle

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis, is an exogenous, B lymphotropic retrovirus belonging to the Retroviridae family that induces persistent lymphocytosis in cattle and sheep. PCR has proven to be particularly suitable for investigating herds of cattle with a very low incidence of BLV infection and for clarifying doubtful serological results obtained by immunodiffusion or ELISA. The native Iranian and Russian cattle have a series of valuable traits that discriminate them as unique breeds that are well able to compete with western analogues. However, their gene pools have not been analyzed with molecular markers, including detection of BLV by PCR. Two pairs of primers were used: gag1 and gag2, and pol1 and pol2, which encompass 347- and 599-bp fragments of the BLV gene, respectively. Sixty-five Iranian Sistani, 120 Yaroslavl, 50 Mongolian, and 35 Black Pied cows were investigated. Among these 270 animals, we obtained 42 positive and 15 doubtful results in the first PCR. The second PCR was very effective in increasing BLV test reliability data to support detection of BLV.     

Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins.

The mechanism of leukemogenesis and persistent lymphocytosis (PL; benign expansion of B lymphocytes) in cattle infected with bovine leukemia virus (BLV; a retrovirus closely related to human T-cell leukemia virus type 1) is unknown; however, the immune system likely plays an important role in controlling the outcome of infection. In this study, we compared T-cell competence in serologically positive alymphocytotic (AL) animals with T-cell functions in animals with progressive stages of infection, PL and tumor bearing (TB). Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30, and gp51) genes in different disease stages. Lymphocytes from AL cattle recognized an average of three of five recombinant proteins per animal. Expansion of antigen pulsed lymphocytes in interleukin-2 increased protein recognition to almost five per animal. In contrast, lymphocytes from PL and TB animals failed to recognize any BLV recombinant proteins. Short-term T-cell cultures from the PL group expanded in interleukin-2, as well as the PL and TB cells cultured in indomethacin (3 to 6 microg/ml), increased the average of recognized proteins per animal to one. Cells proliferating to BLV antigens were CD4+ T lymphocytes, as shown by cell depletion studies. The positive effect of indomethacin suggests involvement of prostaglandin E2 as a negative regulatory factor in the later stages of disease. Thus, for the first time, advancing stages of BLV infection were correlated with decreased T-cell competence, providing deeper insight into pathogenesis of retroviral infections.     

Ex vivo survival of peripheral blood mononuclear cells in sheep induced by bovine leukemia virus (BLV) mainly occurs in CD5- B cells that express BLV

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis (EBL). In a previous report, we found that in a sheep model, only CD5(-) B cells proliferated clonally, while CD5(+) B cells rapidly decreased when the disease progressed to the lymphoma stage. We demonstrate here that, although both CD5(+) and CD5(-) B cells, but not CD4(+) T, CD8(+) T and gammadeltaTCR(+)T cells, are protected from spontaneous ex vivo apoptosis in sheep infected with wild-type and a mutant BLV that encodes a mutant Tax D247G protein with elevated trans-activation activity, only CD5(-) B cells become the main target for ex vivo survival when the disease proceeds to the persistent lymphocytotic stage, which showed an increased expansion of the CD5(-) B cells. In addition, we identified, by four-color flow cytometric analysis, that in CD5(-) B cells, the apoptotic rates of cells that expressed wild-type and mutant BLV were greatly decreased compared with those of BLV-negative cells. There was only a slight reduction in the apoptotic rates in BLV-positive cells from CD5(+) B cells. In addition, supernatants from peripheral blood mononuclear cell (PBMC) cultures from wild-type- and mutant BLV-infected sheep mainly protected CD5(-) B cells from spontaneous apoptosis. Our results suggest that, although BLV can protect both CD5(+) and CD5(-) B cells from ex vivo apoptosis, the mechanisms accounting for the ex vivo survival between these two B-cell subsets differ. Therefore, it appears that the phenotypic changes in cells that express CD5 at the lymphoma stage could result from a difference in susceptibility to apoptosis in CD5(+) and CD5(-) B cells in BLV-infected sheep.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Evaluation of radioimmunoprecipitation for the detection of bovine leukemia virus infection in domestic cattle.

The potential application of a recently developed radioimmunoprecipitation test for antibody directed against the major structural protein of bovine leukemia virus was evaluated for use in detection of BLV-infection in domestic cattle. This technique was found to be considerably more sensitive than serologic procedures currently being utilized for this purpose. Radioimmunoprecipitation was also shown to have distinct advantages as compared to hematologic criteria, such as specified by Bendixen's index, for identification of BLV-infected animals. By the use of radioimmunoprecipitation, high levels of antibody to BLV were demonstrated in sera of animals with confirmed adult lymphosarcoma, but not in animals with a less common sporadic form of the disease which occurs in calves.     

Protection of rats against pseudorabies virus infection by gamma-interferon

Administration of recombinant rat gamma-interferon to rats conferred complete protection against an otherwise lethal intraperitoneal pseudorabies virus (PRV) infection. The primary target cell of the virus has been identified as the serosal cell of the peritoneum. Histologic examination showed that after infection of the underlying adventitia, the virus replicates in the myenteric and submucosal plexuses of the gastrointestinal tract; this is followed by centripetal spread to the autonomous and central nervous system. In recombinant rat gamma-interferon-treated rats, viral antigen was absent in the primary target cells and was not detected in any other organ. In interferon-treated cultures of peritoneal fibroblasts, which represent another primary target cell population in vivo, complete inhibition of PRV replication was observed. The peritoneal macrophage is not susceptible to PRV, as was shown by coculture and immunocytochemical studies. Peritoneal cells from gamma-interferon-treated rats showed enhanced major histocompatibility class II antigen expression and extrinsic antiviral activity in PRV-susceptible rat embryo fibroblasts. The results presented in this study indicate that protection by the lymphokine is likely to be based on direct inhibition of viral replication in serosal cells.     

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.