Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential.

BACKGROUND: Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication. RESULTS: We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV. CONCLUSIONS: Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.

Immunopathologic Effects of Prednisolone and Cyclosporine A on Feline Immunodeficiency Virus Replication and Persistence.

Feline immunodeficiency virus (FIV) induces opportunistic disease in chronically infected cats, and both prednisolone and cyclosporine A (CsA) are clinically used to treat complications such as lymphoma and stomatitis. However, the impact of these compounds on FIV infection are still unknown and understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective therapies. To determine the immunologic and virologic effects of prednisolone and CsA during FIV infection, FIV-positive cats were administered immunosuppressive doses of prednisolone (2 mg/kg) or CsA (5 mg/kg). Both prednisolone and CsA induced acute and transient increases in FIV DNA and RNA loads as detected by quantitative PCR. Changes in the proportion of lymphocyte immunophenotypes were also observed between FIV-infected and naïve cats treated with CsA and prednisolone, and both treatments caused acute increases in CD4+ lymphocytes that correlated with increased FIV RNA. CsA and prednisolone also produced alterations in cytokine expression that favored a shift toward a Th2 response. Pre-treatment with CsA slightly enhanced the efficacy of antiretroviral therapy but did not enhance clearance of FIV. Results highlight the potential for drug-induced perturbation of FIV infection and underscore the need for more information regarding immunopathologic consequences of therapeutic agents on concurrent viral infections.

Mucosal Immune Response to Feline Enteric Coronavirus Infection.

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into naïve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFNγ responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal.

FIV vaccine with receptor epitopes results in neutralizing antibodies but does not confer resistance to challenge.

Feline immunodeficiency virus (FIV) is the feline analogue to human immunodeficiency virus (HIV) and utilizes parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important target for induction of neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block infection ex vivo; thus highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. To determine whether vaccination with CD134-SU complexes could induce protection against FIV infection, cats (n = 5 per group) were immunized with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes. Two trials were performed with different antigen combinations and vaccination schedules. In vivo generation of anti-CD134 and anti-SU IgG antibodies was measured, and in vitro neutralization assays were conducted. Immunization induced production of anti-CD134 and anti-SU antibodies that significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection, and neat serum from vaccinated cats enhanced FIV growth in vitro. CD134+SU vaccinated cats exhibited increased CD4:CD8 ratio immediately prior to challenge, and antibodies were much more efficiently generated against vaccine by-products versus target antigens. Results suggest vaccination against viral and cryptic receptor epitopes yields neutralizing antibodies that synergistically inhibit FIV infection in vitro. Factors contributing to vaccine failure may include: (1) Heat-labile serum factors that enhance viral replication, (2) changes in circulating target cell populations induced by vaccination, and (3) weak immunogenicity of neutralizing epitopes compared to off-target vaccine components. Results reinforce the need to monitor vaccine preparation components and avoid non-specific immune stimulation during vaccination.

Effects of Low-level Brodifacoum Exposure on the Feline Immune Response.

Anticoagulant rodenticides have been implicated as a potential inciting factor in the development of mange in wild felids, but a causative association between anticoagulant rodenticide exposure and immune suppression has not been established. Specific-pathogen-free domestic cats were exposed to brodifacoum over a 6-week period to determine whether chronic, low-level exposure altered the feline immune response. Cats were vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses. Measures of immune response included delayed-type hypersensitivity tests and cell proliferation assays. IgE and antigen-specific antibodies were quantified via ELISA assays, and cytokine induction following exposure to vaccine antigens was also analyzed. While cats had marked levels of brodifacoum present in blood during the study, no cats developed coagulopathies or hematologic abnormalities. Brodifacoum-exposed cats had transient, statistically significant decreases in the production of certain cytokines, but all other measures of immune function remained unaffected throughout the study period. This study indicates that cats may be more resistant to clinical effects of brodifacoum exposure than other species and suggests that the gross impacts of environmentally realistic brodifacoum exposure on humoral and cell-mediated immunity against foreign antigen exposures in domestic cats are minimal.