Vaccination with vif-deleted feline immunodeficiency virus provirus, GM-CSF, and TNF-alpha plasmids preserves global CD4 T lymphocyte function after challenge with FIV

Abstract

Feline immunodeficiency virus (FIV) DNA vaccine approaches that included a vif-deleted FIV provirus (FIV-pPPRDeltavif) and feline cytokine expression plasmids were tested for immunogenicity and efficacy by immunization of specific pathogen free cats. Vaccine protocols included FIV-pPPRDeltavif plasmid alone; a combination of FIV-pPPRDeltavif DNA and feline granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha expression plasmids; or a combination of FIV-pPPRDeltavif and feline interleukin (IL)-15 plasmids. Cats immunized with FIV-pPPRDeltavif, GM-CSF and TNF-alpha plasmids demonstrated an increased frequency of FIV-specific T cell proliferation responses compared to other vaccine groups. Immunization with FIV-pPPRDeltavif and IL-15 plasmids was distinguished from other vaccine protocols by the induction of antiviral antibodies. Suppression of virus loads was not observed for any of the FIV-pPPRDeltavif DNA vaccine protocols after challenge with the FIV-PPR isolate. However, prior immunization with FIV-pPPRDeltavif, GM-CSF, and TNF-alpha plasmids resulted in preservation of CD4 T cell functions, including mitogen-induced cytokine expression and antigen-specific proliferation upon infection with FIV. These findings justify further examination of cytokine combinations as adjuvants for lentiviral DNA vaccines.

Figure 1. Expression of recombinant feGM-CSF in mammalian cells by Western blot analysis

Cell lysates (lane 1) and concentrated cell culture supernatants (lane 3) prepared from COS-7 cells transfected with pCDNA3-feGMCSF were compared to lysates (lane 2) and supernatants (lane 4) prepared from COS-7 cells transfected with control plasmid pCDNA3-rfeGMCSF by immunoblot analysis. The blot was probed with goat polyclonal antibody against feGM-CSF. Arrows indicate two GM-CSF proteins of slightly different sizes, probably due to post-translational modification. The smaller form of feGM-CSF appears as the predominant form in cell lysates and the larger form representing mature secreted glycosylated protein is the predominant form in transfected-cell culture supernatants.

Figure 2. Detection of FIV-specific lymphoproliferative responses after vaccination

PBMC from vaccinated animals were tested for FIV-specific proliferation using a flow cytometric assay based on BrdU-uptake with whole killed virus as antigen. Frequency of proliferating PBMC for different vaccine and control groups are shown for time points before and after vaccination. Arrowheads shown below the graphs for vaccine groups describe the time point of booster immunization. A significant proliferation response was defined as a value greater than, or equal to, 2% FIV-specific proliferating T cells (see dashed cut line). Data was not always available for the same time-points for each group after vaccination. Numbers next to legend symbols represent identity of the experimental animal.

Figure 3. Measurement of FIV proviral DNA concentrations in PBMC of immunized animals after challenge with FIV-PPR

PBMC preparations from cats within vaccine and control groups were assayed for FIV proviral DNA concentrations at 0, 4, 8, and 12 weeks after challenge (A). Proviral DNA concentrations were measured using TaqMan real-time PCR with FIV-PPR gag primer and probe sets. Numbers next to legend symbols represent identity of the experimental animal. Median virus loads with standard error bars for each vaccine group at 0, 4, 8, and 12 weeks after challenge are also described (B).

Figure 4. Detection of FIV-specific lymphoproliferative responses after challenge with FIV-PPR

PBMC from vaccinated and unvaccinated animals were tested for FIV-specific proliferation using a flow cytometric assay based on BrdU-uptake with whole killed virus as antigen. Frequency of proliferating PBMC for different vaccine and control groups are shown for time points ranging from 2 to 16 weeks after challenge. A significant proliferation response was defined as a value greater than, or equal to, 2% FIV-specific proliferating T cells (see dashed cut line). Data was not always available for the same time-points for each group after challenge. Numbers next to legend symbols represent identity of the experimental animal.

Figure 5. Development and validation of a feline intracellular staining cytokine flow cytometric assay for expression of feline IL-2 and TNF-α

A representative pseudo-color plot generated by flow cytometry illustrates intracellular TNF-α and IL-2 staining of feline PBMC after stimulation with mitogens PMA and ionomycin (A). The four-color flow cytometric panels show gating strategy and lymphocyte staining by antibodies for feline CD4, feline CD8, human TNF-α, and human IL-2 as described in Methods. Cross reactivity of a human IL-2 monoclonal antibody (clone MQ1-17H12) for feline IL-2 was confirmed by immunoprecipitation and Western blot analysis (B). Immunoprecipitates were prepared from cell lysates and concentrated culture supernatants derived from mitogen-stimulated feline PBMC and simian COS-7 cells, using the human IL-2 monoclonal antibody. Immunoprecipitates prepared from PBMC lysates (lane 3), concentrated PBMC culture supernatants (lane 4), and COS-7 cells (lane 5) were separated on a 18% polyacrylamide gel and assayed for detection of a 15 kD feline IL-2 protein using a goat anti-feline IL-2 polyclonal antibody. Molecular weight standards (lane 1) and an immunoprecipitate from recombinant human IL-2 (AIDS Research and Reference Reagent Program) (lane 2) provided controls for detection of a feline IL-2 protein of the appropriate size.

Figure 6. Cytokine expression profiles of normal, vaccinated FIV-infected, and unvaccinated FIV-infected cats

PBMC were harvested from FIV-infected cats previously vaccinated with FIVΔvif DNA and feGM-CSF and feTNF-α plasmids (group 1) or with feGM-CSF and feTNF-α plasmids alone (control group 4), and from healthy SPF cats. PBMC were tested for cytokine expression after PMA and ionomycin stimulation using an ICS assay. Frequencies are shown as percentages of CD4 (A) and CD8 (B) lymphocytes expressing either TNF-α alone, IL-2 alone, or co-expressing both cytokines after mitogen stimulation. Mean and standard error of four repeated experiments over multiple time points are depicted in each bar graph. Asterisks above bar graphs indicate a significant difference between values, with P values < 0.05 considered significant as determined by the Mann-Whitney U test.