Ellen E. Sparger

Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus

The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. We have examined regulation of LTR-directed gene expression in feline immunodeficiency virus (FIV), a T-lymphocytopathic lentivirus associated with a fatal AIDS-like disease in domestic cats. Two independent virus isolates, designated FIV-Petaluma and FIV-PPR, have been molecularly cloned and show greater than 85% sequence homology. Both clones (termed pF34 and pPPR) produce infectious virus after transfection of permissive feline cells. Basal promoter activity of the LTRs was measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene. Both LTRs were strong promoters in several cell lines, although in some cell lines the pF34 LTR had four- to fivefold higher basal activity than the pPPR LTR. FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-kappa B site resulted in decreased basal activity of the FIV promoter. Mutational analysis also revealed a negative regulatory element. In cotransfection experiments, both pF34 proviral DNA and pPPR proviral DNA appeared to transactivate either the pF34 LTR or the pPPR LTR; however, levels of transactivation were very low. Cotransfection of both LTRs with FIV subgenomic clones containing various viral open reading frames resulted in low level or no transactivation. The LTRs of both FIV clones responded to cell activation signals in human T-lymphoid cells (Jurkat) treated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Promoter function of both FIV LTRs was also enhanced in cells treated with either forskolin, an inducer of intracellular cyclic-AMP (c-AMP), or dibutyryl c-AMP. Analysis of site-specific mutants showed that a potential AP1 site in the U3 domain of the LTR was required for T-cell activation responses mediated by protein kinase C, whereas a putative ATF site was the target for c-AMP-induced responses mediated by protein kinase A. These studies revealed that cellular transcription factors play a significant role in regulation of FIV gene expression.

Feline Immunodeficiency Virus Orf-A Is Required for Virus Particle Formation and Virus Infectivity

ABSTRACT The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.

Current thoughts on feline immunodeficiency virus infection

The feline immunodeficiency virus (FIV) is a novel feline retrovirus of the lentivirus subfamily that is known to induce immunodeficiency disorders in experimentally and naturally infected cats. Recent studies have characterized the stages of infection and specific immune deficits associated with FIV infection. Host cell populations harboring the virus have been better elucidated. Molecular cloning and nucleotide sequence analysis of the genomes of several isolates of FIV indicate sequence variation in the env gene of FIV, as has been found with other lentiviruses. Sequence of the genome has allowed the development of molecular reagents, including FIV DNA probes, polymerase chain reaction primers, and recombinant viral proteins, which have been and will be useful for FIV diagnostics and for pathogenesis studies. Investigations characterizing FIV as an animal model for human AIDS has yielded significant information concerning FIV infection in naturally infected cats and also may yield FIV vaccines and antiviral therapeutics useful for the pet cat population.

Co-immunization with IL-15 enhances cellular immune responses induced by a vif-deleted simian immunodeficiency virus proviral DNA vaccine and confers partial protection against vaginal challenge with SIVmac251

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a valuable animal model for human immunodeficiency virus (HIV)-1 vaccine development. Our laboratory recently described the immunogenicity and limited efficacy of a vif-deleted SIVmac239 proviral DNA (SIV/CMVDelta vif) vaccine. The current report characterizes immunogenicity and efficacy for the SIV/CMVDelta vif proviral DNA vaccine when co-inoculated with an optimized rhesus interleukin (rIL)-15 expression plasmid. Macaques co-inoculated with rIL-15 and SIV/CMVDelta vif proviral plasmids showed significantly improved SIV-specific CD8 T cell immunity characterized by increased IFN-gamma ELISPOT and polyfunctional CD8 T cell responses. Furthermore, these animals demonstrated a sustained suppression of plasma virus loads after multiple low dose vaginal challenges with pathogenic SIVmac251. Importantly, SIV-specific cellular responses were greater in immunized animals compared to unvaccinated controls during the initial 12 weeks after challenge. Taken together, these findings support the use of IL-15 as an adjuvant in prophylactic anti-HIV vaccine strategies.

Blocking Indolamine-2,3-Dioxygenase Rebound Immune Suppression Boosts Antitumor Effects of Radio-Immunotherapy in Murine Models and Spontaneous Canine Malignancies

Purpose: Previous studies demonstrate that intratumoral CpG immunotherapy in combination with radiotherapy acts as an in-situ vaccine inducing antitumor immune responses capable of eradicating systemic disease. Unfortunately, most patients fail to respond. We hypothesized that immunotherapy can paradoxically upregulate immunosuppressive pathways, a phenomenon we term “rebound immune suppression,” limiting clinical responses. We further hypothesized that the immunosuppressive enzyme indolamine-2,3-dioxygenase (IDO) is a mechanism of rebound immune suppression and that IDO blockade would improve immunotherapy efficacy. Experimental Design: We examined the efficacy and immunologic effects of a novel triple therapy consisting of local radiotherapy, intratumoral CpG, and systemic IDO blockade in murine models and a pilot canine clinical trial. Results: In murine models, we observed marked increase in intratumoral IDO expression after treatment with radiotherapy, CpG, or other immunotherapies. The addition of IDO blockade to radiotherapy + CpG decreased IDO activity, reduced tumor growth, and reduced immunosuppressive factors, such as regulatory T cells in the tumor microenvironment. This triple combination induced systemic antitumor effects, decreasing metastases, and improving survival in a CD8+ T-cell–dependent manner. We evaluated this novel triple therapy in a canine clinical trial, because spontaneous canine malignancies closely reflect human cancer. Mirroring our mouse studies, the therapy was well tolerated, reduced intratumoral immunosuppression, and induced robust systemic antitumor effects. Conclusions: These results suggest that IDO maintains immune suppression in the tumor after therapy, and IDO blockade promotes a local antitumor immune response with systemic consequences. The efficacy and limited toxicity of this strategy are attractive for clinical translation. Clin Cancer Res; 22(17); 4328–40. ©2016 AACR.

Vaccination of Cats with Attenuated Feline Immunodeficiency Virus Proviral DNA Vaccine Expressing Gamma Interferon

ABSTRACT A feline immunodeficiency virus (FIV) provirus with a vif gene deletion (FIVΔvifATGγ) that coexpresses feline gamma interferon (IFN-γ) was tested as a proviral DNA vaccine to extend previous studies showing efficacy with an FIV-pPPRΔvif DNA vaccine. Cats were vaccinated with either FIVΔvifATGγ or FIV-pPPRΔvif proviral plasmid DNA or with both FIV-pPPRΔvif DNA and a feline IFN-γ expression plasmid (pCDNA-IFNγ). A higher frequency of FIV-specific T-cell proliferation responses was observed in cats immunized with either FIVΔvifATGγ or FIV-pPPRΔvif plus pCDNA-IFNγ, while virus-specific cytotoxic-T-lymphocyte responses were comparable between vaccine groups. Antiviral antibodies were not observed postvaccination. Virus-specific cellular and humoral responses were similar between vaccine groups after challenge with a biological FIV isolate (FIV-PPR) at 13 weeks postimmunization. All vaccinated and unvaccinated cats were infected after FIV-PPR challenge and exhibited similar plasma virus loads. Accordingly, inclusion of plasmids containing IFN-γ did not enhance the efficacy of FIV-pPPRΔvif DNA immunization. Interestingly, the lack of protection associated with FIV-pPPRΔvif DNA immunization contrasted with findings from a previous study and suggested that multiple factors, including timing of FIV-pPPRΔvif inoculations and challenge, as well as route of challenge virus delivery, may significantly impact vaccine efficacy.

FIV as a Model for HIV: An Overview

Animal models for human immunodeficiency virus (HIV) infection play a key role in understanding the pathogenesis of AIDS and the development of therapeutic agents and vaccines. As the only lentivirus that causes an immunodeficiency resembling that of HIV infection, in its natural host, feline immunodeficiency virus (FIV) has been a unique and powerful model for AIDS research. FIV was first described in 1987 by Niels Pedersen and co-workers as the causative agent for a fatal immunodeficiency syndrome observed in cats housed in a cattery in Petaluma, California. Since this landmark observation, multiple studies have shown that natural and experimental infection of cats with biological isolates of FIV produces an AIDS syndrome very similar in pathogenesis to that observed for human AIDS. FIV infection induces an acute viremia associated with Tcell alterations including depressed CD4 :CD8 T-cell ratios and CD4 T-cell depletion, peripheral lymphadenopathy, and neutropenia. In later stages of FIV infection, the host suffers from chronic persistent infections that are typically self-limiting in an immunocompetent host, as well as opportunistic infections, chronic diarrhea and wasting, blood dyscracias, significant CD4 T-cell depletion, neurologic disorders, and B-cell lymphomas. Importantly, chronic FIV infection induces a progressive lymphoid and CD4 T-cell depletion in the infected cat. The primary mode of natural FIV transmission appears to be blood-borne facilitated by fighting and biting. However, experimental infection through transmucosal routes (rectal and vaginal mucosa and perinatal) have been well documented for specific FIV isolates. Accordingly, FIV disease pathogenesis exhibits striking similarities to that described for HIV-1 infection.

Construction and In Vitro Characterization of Attenuated Feline Immunodeficiency Virus Long Terminal Repeat Mutant Viruses

ABSTRACT AP-1- and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiplecis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines.

Canine cancer immunotherapy studies: linking mouse and human

Despite recent major clinical breakthroughs in human cancer immunotherapy including the use of checkpoint inhibitors and engineered T cells, important challenges remain, including determining the sub-populations of patients who will respond and who will experience at times significant toxicities. Although advances in cancer immunotherapy depend on preclinical testing, the majority of in-vivo testing currently relies on genetically identical inbred mouse models which, while offering critical insights regarding efficacy and mechanism of action, also vastly underrepresent the heterogeneity and complex interplay of human immune cells and cancers. Additionally, laboratory mice uncommonly develop spontaneous tumors, are housed under specific-pathogen free conditions which markedly impacts immune development, and incompletely model key aspects of the tumor/immune microenvironment. The canine model represents a powerful tool in cancer immunotherapy research as an important link between murine models and human clinical studies. Dogs represent an attractive outbred combination of companion animals that experience spontaneous cancer development in the setting of an intact immune system. This allows for study of complex immune interactions during the course of treatment while also directly addressing long-term efficacy and toxicity of cancer immunotherapies. However, immune dissection requires access to robust and validated immune assays and reagents as well as appropriate numbers for statistical evaluation. Canine studies will need further optimization of these important mechanistic tools for this model to fulfill its promise as a model for immunotherapy. This review aims to discuss the canine model in the context of existing preclinical cancer immunotherapy models to evaluate both its advantages and limitations, as well as highlighting its growth as a powerful tool in the burgeoning field of both human and veterinary immunotherapy.

Transcriptional Regulation of Latent Feline Immunodeficiency Virus in Peripheral CD4+ T-lymphocytes

Feline immunodeficiency virus (FIV), the lentivirus of domestic cats responsible for feline AIDS, establishes a latent infection in peripheral blood CD4+ T-cells approximately eight months after experimental inoculation. In this study, cats experimentally infected with the FIV-C strain in the asymptomatic phase demonstrated an estimated viral load of 1 infected cell per approximately 103 CD4+ T-cells, with about 1 copy of viral DNA per cell. Approximately 1 in 10 proviral copies was capable of transcription in the asymptomatic phase. The latent FIV proviral promoter was associated with deacetylated, methylated histones, which is consistent with a condensed chromatin structure. In contrast, the transcriptionally active FIV promoter was associated with histone acetylation and demethylation. In addition, RNA polymerase II appeared to be paused on the latent viral promoter, and short promoter-proximal transcripts were detected. Our findings for the FIV promoter in infected cats are similar to results obtained in studies of human immunodeficiency virus (HIV)-1 latent proviruses in cell culture in vitro studies. Thus, the FIV/cat model may offer insights into in vivo mechanisms of HIV latency and provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus.