Appearance of cytotoxic antibody to viral gp70 on feline lymphoma cells (FL-74) in cats during ex vivo immunoadsorption therapy: quantitation, characterization, and association with remission of disease and disappearance of viremia.

Fifty cats with feline leukemia virus (FeLV) infection and leukemia-lymphoma complex were treated by ex vivo immunoadsorption with Staphylococcus protein A-bound filters. Most cats responded to therapy. Twelve showed tumor regression, including disappearance of tumor cells, but died later of other complications. Three have had long-term remission of more than 1 year and remain healthy. A consistent finding in these three cats was the appearance during treatment of a complement-dependent cytotoxic antibody against cat lymphoma cells (FL-74). The cytotoxic antibody increased substantially during treatment. Appearance and increase of the cytotoxic antibody was associated with disappearance of FeLV from blood and remission of leukemia. By electroblot analysis, antibody to FeLV protein (Mr, 70,000) was detected in serum prior to detection of the cytotoxic antibody. The cytotoxic antibody was found by immunofluorescence to be specific for antigens on membranes of viable FL-74 cells. By using monoclonal antibodies to FL-74 cells and to components of FeLV, the cytotoxic antibody was shown to be directed against gp70, a glycoprotein of Mr 70,000, but not against p27 of FeLV or other membrane antigen(s) of FL-74 cells. The development of a high concentration of cytotoxic antibody to FeLV gp70 may play an important role in tumor regression and in disappearance of FeLV infection.     

Effects of tumor necrosis factor-alpha on normal feline hematopoietic progenitor cells.

The effects of tumor necrosis factor-alpha (TNF-alpha) on feline bone marrow hematopoietic progenitors were evaluated by exposing bone marrow mononuclear cells from specific pathogen-free cats to different concentrations of TNF-alpha (ranging from 50 to 800 pg/ml) for 2 h before plating for clonal assays of colony-forming units. TNF-alpha caused a dose-dependent suppression of feline erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E), whereas granulocyte-macrophage colony-forming units (CFU-GM) were minimally affected. TNF-alpha concentrations as low as 200 pg/ml significantly inhibited growth of erythroid progenitors. Addition of polyclonal rabbit anti-TNF-alpha antibodies completely neutralized the suppressive effect of TNF-alpha on erythroid progenitors. At higher concentrations of TNF-alpha (800 pg/ml), 35% of CFU-E and 21% of BFU-E still survived, indicating that some erythroid progenitors are not sensitive to a single exposure of TNF-alpha in vitro. These results suggest that TNF-alpha may play a role in regulating hematopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukemia virus.     

In vivo infection of marrow stromal fibroblasts by feline leukemia virus.

Marrow stromal fibroblasts (FBs) likely play an important role in the regulation of hematopoiesis within the marrow microenvironment. Infection of these cells by feline leukemia virus (FeLV) might not only contribute to the pathogenesis of FeLV-induced hematologic diseases, but could provide a reservoir for virus in the infected cat. To determine the frequency of FeLV infection among marrow FB precursor cells (fibroblast colony-forming units, CFU-F) of cats viremic with FeLV-C/Sarma and FeLV-A/61E, marrow FBs and FB cell clones were isolated and assayed for expression of FeLV gag protein. From 30% to 86% and 64% to 88% of marrow FB precursors were infected with FeLV-C/Sarma and FeLV-A/61E, respectively. CFU-F from a cat viremic with FeLV-A/61E were not affected by exposure to antibody against FeLV envelope glycoprotein gp70 and heterologous complement, whereas similarly treated hematopoietic progenitors (erythroid colony-forming units, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte-macrophage colony-forming units, CFU-GM) and culture-propagated, FeLV-infected marrow FBs were effectively lysed, suggesting that infected CFU-F within the marrow microenvironment do not express a significant amount of gp70 on their cell membranes. Thus, marrow FB precursor cells appear to be a major target for FeLV in vivo. Furthermore, the low level of gp70 antigen expression on the surface of these cells in vivo may allow them to escape immune surveillance and provide a reservoir of virus during active or latent infection.     

Induction of feline immunodeficiency syndrome by feline leukemia virus: pituitary and adrenocortical dysfunctions

Seven young cats were injected with feline leukemia virus (FeLV); six of them became viremic. All of the viremic cats developed AIDS-related symptoms, i.e. lymphopenia, neutropenia, thymic atrophy, and wasting syndrome, along with an altered pituitary and adrenocortical function. These symptoms closely resemble human AIDS induced by HIV. It was discovered that, after 2 weeks of infection, the average amount of plasma adrenocorticotropic hormone (ACTH) detected in the infected cats was reduced by 29% in comparison with that before the infection. In contrast to the second week, the fifth week of infection showed a 94% increase of plasma ACTH which then dropped back down to 38% after the sixth and seventh weeks. This opposing biphasic pattern of change was also observed in the plasma cortisol content of the infected cats. The amount of change in plasma cortisol did not correlate with the detected increase in plasma ACTH, indicating a weak adrenal response to pituitary action.     

Retrovirus-encoded transformation-specific polyproteins: expression coordinated with malignant phenotype in cells from different germ layers

A transformation-associated polyprotein designated "gag-x" was previously shown to be induced by the feline sarcoma virus (FeSV) after the nonproductive transformation of rat or mink cells. We found that this protein was also expressed in cells derived from the native species (cat) with or without the production of feline leukemia helper virus (FeLV) and that cats could mount a humoral antibody response to the transformation-specific (x) portion of the molecule. Such antisera also reacted with the feline oncornavirus-associated cell membrane antigen (FOCMA) by membrane immunofluorescence. Expression of the gag-x protein was coordinated with malignant phenotype in that both transformed cat fibroblasts and cultured cells from a FeSV-induced melanoma expressed antigenically indistinguishable proteins of the same size. These cells are derived from different embryonic germ layers, suggesting that such transformation-related proteins may function in a pleiotropic manner when introduced by a virus.     

Improved health and survival of FIV-infected cats is associated with the presence of autoantibodies to the primary receptor,CD134

We analyzed antibody responses in sera from feline immunodeficiency virus (FIV)-infected and uninfected cats. A strong antiviral response to the viral surface glycoprotein (SU) was noted in both natural and experimental infections. In addition, 143 of 226 FIV-infected animals (63%) also expressed antibodies to the primary binding receptor, CD134, whereas cats infected with other feline RNA viruses, including calicivirus, coronavirus, herpesvirus, and feline leukemia virus, did not. Both affinity-purified anti-CD134 and anti-SU antibodies blocked FIV infection ex vivo. FACS analyses revealed that the anti-CD134 antibodies bound to a cryptic epitope on the receptor that was only exposed when SU bound to CD134. Anti-CD134 binding caused displacement of SU from the surface of the cell and inhibition of infection. The presence of antibodies to CD134 correlated with lower virus loads and a better overall health status in FIV⁺ cats, whereas anti-SU antibodies were present independent of health status. The findings are consistent with a role for antireceptor antibodies in protection from virus spread and disease progression.     

Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses.

Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effects on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These findings suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.     

Bone marrow colony-forming unit assay in cats with naturally occurring myelodysplastic syndromes

Feline myelodysplastic syndromes (MDS) has been diagnosed in many cats infected with feline leukemia virus, although the pathogenesis of this hematopoietic deficiency has been unclear. In this study, we assayed the bone marrow erythroid colony-forming units (CFU-E) and granulocyte-machrophage CFUs (CFU-GM) to investigate the pathogenesis of feline MDS. The number of CFU-E colonies was decreased in 4 of 7 cats with MDS, and the number of CFU-GM colonies was also decreased in 4 cats. Furthermore, small colonies of CFU-GM were found in all 7 cases. These findings indicated that refractory cytopenia of feline MDS could be caused by abnormal maturation and differentiation of hematopoietic stem cells in bone marrow, as it is in human MDS. The pathogenesis of feline MDS might be similar to that of human MDS.     

Common Cell-Surface Antigen Associated with Murine and Feline C-Type RNA Leukemia Viruses

A new common cell-surface antigen associated with murine and feline C-type RNA leukemia viruses was demonstrated by the use of rabbit antiserum against feline leukemia virus and the indirect membrane immunofluorescence test. Common cell-surface antigen was found in all leukemias of all strains of mice tested, in normal lymphoid tissues of Gross-positive (high incidence of leukemia) mouse strains AKR, AKR.H-2(b), C58, and NZB, in cultured rat fibroblasts infected with Rauscher virus, in cultured feline fibroblasts infected with feline leukemia virus, and in spontaneous feline lymphosarcoma. The antigen was not demonstrable in normal adult and fetal tissues of Gross-negative mouse strains or in tissues and cultured fibroblasts derived from normal rats and normal cats. The immunoferritin study of murine leukemia cells revealed that the antigen was located on the cell surface in discrete areas; budding and C-type RNA viral envelope was not labeled as antigen site. The distribution of common cell-surface antigen on murine and feline leukemias, as well as on normal lymphoid tissues of Gross-positive mouse strains, indicates the presence of an antigen distinct from any cell-surface antigen heretofore shown to be associated with, or specified by, mammalian C-type RNA viruses.     

Immune response of healthy and leukemic cats to the feline oncornavirus-associated cell membrane antigen.

Feline leukemia is an infectious disease caused by a horizontally transmitted virus. Infection of animals or cultured cells with feline oncornaviruses results in the expression of a specific cell membrane antigen, feline oncornavirus-associated cell membrane antigen (FOCMA). The humoral antibody response to FOCMA is directly correlated with tumor progression. The measurement of this antibody is a useful tool for determining virus exposure. Using this procedure it was determined that cats living in leukemia "cluster" households as well as cats used as contact controls in virus injection experiments have a risk of infection of 90% or higher.     

Horizontal transmission of feline leukemia virus in cats.

Traditionally, cancer has not been considered an infectious disease although some multiple cases of leukemia in man and cattle have been reported. The discovery that feline lymphosarcoma was associated with an RNA virus (feline leukemia virus(FeLV)) meant that infectious transmission of the disease was a possibility. The critical question was whether the predominant method of transmission from one animal to another was 'vertical' (via the gametes) or 'horizontal' (via contagion or infection). A number of epidemiological studies have shown that the chances of healthy cats contracting lymphosarcoma are greatly increased when a cat with the disease lives in close proximity. It does not matter whether the healthy cats are related to the sick animal or not. It has also been established that viremic normal cats have an approximately 900 times greater chance of developing leukemia than cats whose FeLV status is unknown. Infectious FeLV is present in the excretions and blood of viremic animals. In the natural environment, feline lymphosarcoma occurs in clusters. The results in pet cats have been supported by experiments with cat colonies under controlled conditions and prove that horizontal transmission of FeLV occurs. This does not mean that epigenetic (infection in utero or via the milk) or vertical transmission cannot also occur. It should be possible to break the cycle of horizontal transmission of the virus by vaccination and thus control FeLV-related diseases.     

Trans-Species Rescue of Defective Genomes of Murine Sarcoma Virus from Hamster Tumor Cells with Helper Feline Leukemia Virus

This report describes a trans-species rescue of defective MSV genome with helper leukemia virus derived from cats. This rescue was achieved by in vitro co-cultivation of hamster tumor cells with feline embryo cells in the presence of helper feline leukemia virus (FeLV), or by inoculation of tumor cells into FeLV-infected newborn cats.The rescued focus-forming viruses produced foci in feline embryo cultures but not in cultures of mouse, rat, and hamster species. One isolate was tested and found to induce sarcoma in a kitten. Antigenic and viral interference studies indicated that the focus-forming virus has the viral envelope of FeLV. Virus stocks consisted of a mixture of focus-forming particles and a 1000-fold excess of helper FeLV. Virus assay pattern in feline embryo cultures with or without added helper FeLV indicated that this helper virus is required for the transformation of feline cells.     

Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta-galactosidase gene of phage lambda Charon 16 so as to obtain expression of random protein fragments as fusion proteins. The monoclonal antibody is used to screen the phage library to isolate phage expressing the specific antigenic determinant. DNA of immunoreactive phage can be analyzed rapidly and subcloned to allow DNA sequence determination. The method is generally applicable and permits antigenic determinants of functionally interesting monoclonal antibodies to be mapped and related to specific protein sequences. We have used this procedure to determine the region of the feline leukemia virus envelope protein gp70 recognized by a virus-neutralizing monoclonal antibody, cl.25. Antibody binding was mapped to a 14-amino acid region in the amino-terminal half of gp70. This region may be directly involved in an essential function of the gp70 protein, perhaps in gp70-mediated host recognition functions. Synthetic peptides derived from this region may provide useful vaccine antigens for the prevention of feline leukemia virus-associated disease in cats.     

Phase I-II study of continuous-infusion high-dose human lymphoblastoid interferon and the in vitro sensitivity of leukemic progenitors in nonlymphocytic leukemia

Twelve pediatric patients with nonlymphocytic leukemia were treated for 10 days with high-dose (15, 20, or 30 million U/m2/day) human lymphoblastoid interferon (Wellferon) administered by continuous iv infusion. Nine children had acute nonlymphocytic leukemia (ANLL) in relapse, two had Philadelphia chromosome-positive chronic myelocytic leukemia in myeloblastic crisis, and one had juvenile chronic myelocytic leukemia. Blast cell counts in the peripheral blood decreased in five patients with ANLL treated with the higher interferon doses; however, there was no evidence of an antileukemic effect in the marrow. Dose-limiting toxicity, which included malaise, hepatotoxicity, and coagulation abnormalities, was observed in patients given 20 or 30 million U/m2/day. Studies of the growth of leukemic progenitor cells in vitro in the presence of interferon disclosed a concentration-related inhibition of colony formation. Patients who had a decrease in peripheral blast cell counts demonstrated greater in vitro inhibition of clonogenic leukemic progenitors than patients whose blast cell counts did not decrease. However, the serum interferon concentrations in patients given clinically tolerable doses were lower than those concentrations which inhibited leukemic cell growth in vitro by a median of 42% (1000 U/ml). This study failed to demonstrate clinically significant antileukemic activity against nonlymphocytic leukemia in patients given high-dose constant-infusion interferon, and the toxicity of this approach was prohibitive.     

Productive infection of T-helper lymphocytes with feline immunodeficiency virus is accompanied by reduced expression of CD4.

An antigen-specific feline T-lymphocyte cell line (Q201) was generated and infected in vitro with the feline immunodeficiency virus (FIV). Syncytium formation and the release of the viral core protein p24 into culture fluid were accompanied by a reduction in expression of the CD4 surface antigen. The reduction in CD4 expression was transient, the resulting persistently infected population of cells expressing levels of CD4 comparable to those observed prior to infection. Persistently infected cells gradually lost expression of major histocompatibility antigen (MHC) class II while maintaining pre-infection levels of expression of CD4, MHC class I, CD18 or CD29.     

Protection against FIV challenge infection by genetic vaccination using minimalistic DNA constructs for FIV env gene and feline IL-12 expression

OBJECTIVE: To evaluate the efficacy of a genetic vaccination protocol based on minimalistic, immunogenic defined gene expression (MIDGE) vectors coding for domains of the feline immunodeficiency virus (FIV) env gene and feline IL-12. METHODS: Three groups of four cats each were immunized three times within 6 weeks by the ballistic transfer of gold particles coated with MIDGE vectors. Group 1 received non-coated gold beads, groups 2 and 3 MIDGE vectors expressing FIV surface plus part of the transmembrane protein. In addition, group 3 received feline IL-12 DNA. All cats were challenged by intraperitoneal injection of 25 TCID50 of infectious FIV Z2. The following criteria were monitored: clinical signs, antibodies to transmembrane protein, antibodies to whole FIV, haematological parameters and kinetics of CD4 and CD8 cells, FIV proviral load (determined by quantitative polymerase chain reaction; PCR) and cytotoxic T lymphocyte (CTL) activity (in selected cats). RESULTS: None of the cats developed a detectable antibody response during immunizations. Four weeks after challenge exposure, all cats in group 1 (control) and group 2 (FIV surface-transmembrane protein) had seroconverted and showed a high proviral load until week 19 (end of experiment). In contrast, only one of four cats in group 3 (surface-transmembrane protein and IL-12) showed antibodies; it was provirus positive at reduced virus load. Short-lived CTL activity was found in two cats in group 3. CONCLUSION: Genetic vaccination using a MIDGE-based construct for the expression of the surface-transmembrane protein domain of FIV env and feline IL-12 DNA led to protection against homologous virus challenge in three out of four vaccinated cats.     

Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.     

Circulating antibodies to the feline leukemia virus and FOCMA in cats

Presence of antibodies to the structural proteins of the feline leukemia virus and the feline oncornavirus-associated cell membrane antigen FOCMA was analyzed in sera of 120 cats from household environments. In our experiments 35% of the sera tested were positive with the viable cells of FL-74 feline lymphoma cell line. On the other hand, only 5% of the feline sera reacted with FeLV from FL-74 cells in the solid phase RIA. Selected positive sera were analyzed for the presence of antibodies to the viral structural proteins of FeLV and FOCMA in lysates of FL-74 cells by radioimmunoprecipitation. Absorption of the selected sera (reacting with viable FL-74 cells) with disrupted FeLV had a negligible effect on the precipitating activity of the 70000 protein.