Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains.

cDNAs encoding four different canine immunoglobulin G (caIgG) gamma chains were identified in this study. One of these IgG gamma chain cDNAs, (caIgG-A), represents 92.5% of the IgG gamma chain cDNAs in a dog spleen cell cDNA library; a second partial IgG gamma chain cDNA (caIgG-B) was also identified in the library. The other two IgG gamma chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG gamma chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG gamma chains, the hinge regions were distinct, sharing only 19.0-35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG gamma chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy gamma chains (A, B, C and D), respectively. Specific primers were designed based on caIgG gamma chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG gamma chain mRNA levels in canine PBMC samples.

Rearranged T lymphocyte antigen receptor genes as markers of malignant T cells.

We have recently cloned a number of canine T cell receptor (TCR) Vbeta genes using degenerate oligonucleotides. From the DNA sequences of the resulting clones and the canine Vbeta gene sequences in the literature, seven distinct canine TCR Vbeta genes were identified. Vbeta specific PCR primers were designed for each of the seven TCR Vbeta genes such that under defined conditions, each primer could only amplify a specific TCR Vbeta gene in conjunction with the same 3' constant region (Cbeta) primer. By performing RT-PCR on RNA derived from a source containing T lymphocytes, the presence and expansion of T cells expressing a particular Vbeta gene could be detected. Moreover, the clonality or diversity of a T cell population under analysis could be easily determined by the VDJ junctional sequence of the amplified Vbeta PCR product, in the form of a "DNA fingerprint". These findings have been used to detect canine T cell lymphoma, and could potentially be used to monitor the remission of T cell malignancies in response to treatment.

Gliding bacterial adjuvant stimulates feline cytokines in vitro and antigen-specific IgG in vivo.

Gliding bacterial adjuvant (GBA) has been previously characterized as a potent immune modulator, stimulating the growth of murine B lymphocytes, inducing murine NK cell activity, and promoting the release of several murine cytokines. Based on these studies and our interest in potentiating the effectiveness of feline vaccines, GBA was tested for its ability to stimulate feline T cells in vitro and act as a vaccine adjuvant in vivo. In vitro, GBA stimulated feline PBLs to proliferate and release interferon (IFN) and IL-2. Unlike IFN, the release of IL-2 appeared to be unaffected by prior depletion of macrophages, indicating GBA directly stimulated feline T cells. In vivo GBA was co-administered with Keyhole Limpet Hemacyanin (KLH) and the anti-KLH antibody response was compared to cats receiving KLH emulsified in complete Freund's adjuvant (CFA). Fourteen days after the third immunization and continuing for a 30-day observation period, KLH-specific IgG titers in cats receiving GBA were significantly higher than those given CFA. However, when cats were subsequently boosted with KLH alone, those cats receiving CFA demonstrated significantly higher antibody titers throughout a second 30-day observation period. The anti-KLH antibody memory response was greatly enhanced when GBA was emulsified with incomplete Freunds adjuvant (IFA) prior to injection. Serum titers of cats given KLH in an oil-based GBA preparation were significantly higher than cats receiving KLH adjuvanted with IFA or CFA, an effect which persisted 38 days after boosting with KLH alone. Finally, GBA significantly enhanced the feline humoral response to a recombinant protein of Dirofilaria immitis, the causative agent of feline heartworm. Serum titers of cats inoculated with recombinant antigen in GBA were significantly greater than cats given recombinant antigen adjuvanted with Titermax, alum, or NAGO. These studies indicate that GBA induces T cell proliferation and the release of IL-2 and IFN in vitro and can be used to enhance the recall antibody response to both a T cell dependent antigen and an immunogen derived from Dirofilaria immitis.

Development of monoclonal antibodies and capture immunoassays for feline immunodeficiency virus.

We generated monoclonal antibodies (MAB) against feline immunodeficiency virus (FIV) and characterized these MAB by single competition enzyme immunoassays (EIA), immunoblot analysis, and radioimmunoprecipitation. Four MAB identified 3 distinct epitopes of the FIV p24/26 gag major core protein. One MAB recognized the p16/17 gag protein; none recognized envelope proteins. We developed an FIV p26 antigen capture EIA that proved more sensitive (0.5 ng of p26/ml), less expensive, and less time-consuming than reverse transcriptase assay. The same MAB were used to develop an antibody EIA specific for FIV p26. The MAB and capture assays reported should prove useful in FIV diagnosis and research.

Early pathogenesis of disease caused by SIVsmmPBj14 molecular clone 1.9 in macaques.

We have studied the early pathogenesis of infection by molecular clone 1.9 of SIVsmmPBj14 in pig-tailed and cynomolgus macaques. Like the uncloned PBj14 parent, SIVsmmPBj14-1.9 consistently induced an acute clinical syndrome characterized by behavioral depression, fever, profuse diarrhea, dehydration, lymphadenopathy, splenomegaly, and mucocutaneous exanthema that began at 7 days postinfection (DPI). The acute clinical disease coincided with a marked cell-associated and cell-free viremia, during which SIV p27 was demonstrated in 4 to 68% of circulating mononuclear leukocytes between 4 and 17 DPI. Also characteristic were monocytosis and reductions in CD4+ and CD8+ T lymphocytes, as well as CD20+ B lymphocytes. The most profound depletion occurred in the CD44hi subset of CD4+ T cells. Unlike animals infected previously with uncloned or biologically cloned PBj14, however, all SIVsmmPBj14-1.9-infected macaques survived the acute-phase disease to progress to a chronic, largely asymptomatic phase of infection. Recovery from the acute-phase disease correlated with down modulation of virus replication and the appearance of antibodies to SIV Env and Gag proteins. Similar to the PBj14 parent, PBj14-1.9 targeted to intestine, spleen, bone marrow, lymph node, and cerebellum. Saliva contained substantial quantities of infectious virus and no viral antibodies during the early phase of infection. By contrast, saliva from chronically infected animals usually contained antibodies but no virus. This study extends previous work demonstrating that the acute clinical syndrome produced by SIVsmmPBj14 in pig-tailed macaques represents a unique model of lentiviral pathogenesis.

Feline immunodeficiency virus neurotropism: evidence that astrocytes and microglia are the primary target cells.

To investigate the neuropathogenesis of feline immunodeficiency virus (FIV) infection in vitro, we have utilized three populations of cultured feline neural cells (astrocytes, microglia, brain endothelium) to assess the relative susceptibility to FIV infection, ability to produce viral antigens, and effects of infection on cell survival. Astrocytes appeared to be the most susceptible to infection, followed by microglia, whereas brain endothelial cells were relatively resistant to infection. Astrocyte infection resulted in syncytium formation and cell death, while microglial cells remained persistently and productively infected, without obvious cytopathic effects. These results suggest that FIV entry into the central nervous system probably does not occur via infected endothelium and that both astrocytes and microglia are more likely target cells for the virus.

Alpha interferon (2b) in combination with zidovudine for the treatment of presymptomatic feline leukemia virus-induced immunodeficiency syndrome.

The therapeutic efficacies of human recombinant alpha interferon (IFN-alpha), IFN-alpha plus zidovudine (AZT), and AZT alone were evaluated in presymptomatic cats with established feline leukemia virus (FeLV)-acquired immunodeficiency syndrome (FAIDS) infection and high levels of persistent antigenemia. Subcutaneous injection of 1.6 x 10(6) U of human recombinant IFN-alpha 2b per kg delivered peak concentrations in plasma of 3,600 U/ml at 2 h postadministration with a half-life of elimination of 2.9 h. This dosage of IFN-alpha could be delivered to cats for up to 12 weeks without significant clinical toxicity. Oral administration of AZT (20 mg/kg three times daily) resulted in peak concentrations in plasma of 3 micrograms/ml at 2 h with a half-life of elimination of approximately 1.60 h. Treatment of FeLV-FAIDS-infected cats with IFN-alpha, either alone or in combination with orally administered AZT, resulted in significant decreases in circulating p27 core antigen beginning 2 weeks after the initiation of therapy. AZT alone had no effect on circulating virus antigen. Depending upon whether high (1.6 x 10(6) U/kg)- or low (1.6 x 10(4) to 1.6 x 10(5) U/kg)-dosage IFN-alpha was used, cats became refractory to therapy 3 or 7 weeks after the beginning of treatment. At these times, IFN-alpha-treated animals developed antibodies to IFN-alpha that were neutralizing, specific for human recombinant IFN-alpha, and dose dependent in magnitude. The results of this study indicate that human recombinant IFN-alpha is effective in reducing circulating virus antigenic load in cats persistently infected with FeLV-FAIDS. However, the continued efficacy of IFN-alpha therapy appeared to be limited by the formation of cytokine-specific neutralizing antibodies.