AIDS vaccination studies with an ex vivo feline immunodeficiency virus model: analysis of the accessory ORF-A protein and DNA as protective immunogens

Abstract

Determining which antigen must be included in AIDS vaccines to confer maximum protection is of utmost importance. In primate models, vaccines consisting of or including accessory viral proteins have yielded conflicting results. We investigated the protective potential of the accessory protein ORF-A of feline immunodeficiency virus (FIV) in cats. All three immunization strategies used (protein alone in alum adjuvant, DNA alone, or DNA prime-protein boost) clearly generated detectable immune responses. Upon challenge with ex vivo homologous FIV, ORF-A-immunized cats showed distinct enhancement of acute-phase infection relative to mock-immunized animals given alum or empty vector DNA. This effect was tentatively attributed to increased expression of the FIV receptor CD134 that was observed in the immunized cats. However, at subsequent sampling points that were continued for up to 10 months postchallenge, the average plasma viral loads of the ORF-A-immunized animals were slightly but consistently reduced relative to those of the control animals. In addition, CD4(+) T lymphocytes in the circulation system declined more slowly in immunized animals than in control animals. These findings support the contention that immunization with lentiviral accessory proteins can improve the host's ability to control virus replication and slow down disease progression but also draw attention to the fact that even simple immunogens that eventually contribute to protective activity can transiently exacerbate subsequent lentiviral infections.

FIG. 1.

Schematic representation of the ORF-A protein with its recognized domains and hydrophobicity profile as calculated with Kyte and Doolittle's algorithm. The nine 15-mer peptides used in the ELISPOT and CTL assays are also shown: as depicted, they encompassed the entire molecule and overlapped by seven residues.

FIG. 2.

ORF-A protein production in E. coli. (A) Time course of IPTG induction evaluated in a polyacrylamide gel with Coomassie blue. (B) Fractions eluted from a Ni-NTA resin column. A sample of each fraction was run in a polyacrylamide gel and analyzed by Coomassie blue staining (top gel) or Western blot with an anti-His monoclonal antibody (bottom gel). The positions of molecular mass markers (in kilodaltons) are indicated to the right of the gels in panels A and B. (C) Fractions eluted from the HR 16/5 column packed with ProBond resin and analyzed in a polyacrylamide gel with Coomassie blue staining. This was the preparation used for cat immunization and immunological tests.

FIG. 3.

ORF-A-specific immune responses in the immunized and mock-immunized cats. Each symbol shows the value from one cat. (A) Antibody response. Plasma samples obtained at the times indicated and diluted 1:100 were examined for ELISA reactivity against H-ORF-A. The dotted line represents the cutoff value calculated by multiplying by 5 the average absorbance (OD₄₀₅) of plasma samples from 10 FIV-naive animals. (B) IFN-γ-secreting T-cell response. At the times indicated, PBMC were stimulated with 5 μM (left symbols) or 1 μM (right symbols) pooled ORF-A oligopeptides (Fig. 1) and directly examined by ELISPOT analysis. (C) ORF-A-specific CTL activity in the PBMC as determined against autologous skin fibroblasts charged with 5 μM of the same pool of ORF-A oligopeptides used in the ELISPOT assay. Shaded areas are means ± standard deviations of background spot-forming cells (B) or background CTL activity detected in the absence of the ORF-A peptides in the assays (C).

FIG. 4.

Outcome of FIV challenge in the immunized and mock-immunized cats. Each symbol shows the value from one cat. (A) Plasma viremia determined by reverse transcription TM-PCR. (B) Proviral loads in the PBMC determined by TM-PCR. (C and D) CD4⁺ and CD8⁺ T lymphocytes in the PBMC. One and two asterisks indicate significant differences relative to the values for the pooled control groups NC and ND at P ≤ 0.05 and P ≤ 0.02, respectively.

FIG. 5.

Postchallenge ORF-A-specific immune responses in the study cats. ORF-A-specific antibodies (A), IFN-γ-secreting cell numbers (B), and CTL activity (C) determined as described in the legend to Fig. 3.

FIG. 6.

FIV receptor (CD134) and coreceptor (CXCR4) gene expression in the PBMC at different times relative to initiation of immunization. Each symbol shows the value for one cat. (A) CD134 (black boxes), CXCR4 (white boxes), and GAPDH (shaded areas) mRNA content in PBMC and determined by TM-PCR and reported as threshold cycle (C_T). (B) Normalized values of individual cats for CD134 and CXCR4, respectively. Data represent changes obtained by subtracting the C_T of GAPDH from the C_T of CD134 or CXCR4 obtained with PBMC collected at the times indicated and dividing by the corresponding value obtained with PBMC collected before immunization was started ( method).