A peptide-loaded dendritic cell based cytotoxic T-lymphocyte (CTL) vaccination strategy using peptides that span SIV Tat, Rev, and Env overlapping reading frames

Abstract

CTL based vaccine strategies in the macaque model of AIDS have shown promise in slowing the progression to disease. However, rapid CTL escape viruses can emerge rendering such vaccination useless. We hypothesized that such escape is made more difficult if the immunizing CTL epitope falls within a region of the virus that has a high density of overlapping reading frames which encode several viral proteins. To test this hypothesis, we immunized macaques using a peptide-loaded dendritic cell approach employing epitopes in the second coding exon of SIV Tat which spans reading frames for both Env and Rev. We report here that autologous dendritic cells, loaded with SIV peptides from Tat, Rev, and Env, induced a distinct cellular immune response measurable ex vivo. However, conclusive in vivo control of a challenge inoculation of SIVmac239 was not observed suggesting that CTL epitopes within densely overlapping reading frames are also subject to escape mutations.

Figure 1

Nucleic acid alignment of the second exon of Tat for all SIVmac strains relative to SIVmac239. Highlighted residues are identical to that of SIVmac239.

Figure 2

IFN-γ T-cell responses against the overlapping epitopes of Tat, Rev, and Env. Using an ELISpot assay, we measured IFN-γ T-cell responses against the peptides used in the vaccination protocol. The vaccinated animals (Panel A), AT56, AT57, AV89, and BA20, each developed strong to moderate responses against every peptide pool tested at a least one time point, except AT56 against Rev pool B. The control animals (Panel B), H405 and T687, did not demonstrate any significant activity throughout the study. The activity levels against the peptide pools, Tat A, Tat B, Rev A, Rev B, Env A, and Env B, are shown for each animal in spot forming cells (SFC) per 10⁶ PBMC. Data are shown from pre-immunization and post-immunization assays. The average numbers of SFC per PBMC and the standard deviations (error bars) were determined from duplicate wells. Responses greater than 50 SFC/10⁶ PBMC were considered positive.

Figure 3

SIVmac239 plasma viremia over time. Plasma samples were measured from the corresponding time points for SIV RNA concentration via the bDNA assay. The data from the vaccinated animals (AT56, AT57, AV89, and BA20) are shown in Panel A, while those from the controls (H405 and T687) are shown in Panel B.

Figure 4

CD4⁺ T-cell counts. Peripheral blood CD4⁺ T-cell counts were longitudinally determined by flow cytometry. The data from the vaccinated animals (AT56, AT57, AV89, and BA20) are shown in Panel A, while those from the controls (H405 and T687) are shown in Panel B.

Figure 5

IFN-γ T-cell responses against Tat, Rev, and Env after SIV infection. IFN-γ T-cell responses of the vaccinated animals were again measured by a IFN-γ ELISpot assay on PBMC from Days 28 and 42 post-infection. The activity levels against the peptide pools, Tat A, Tat B, Rev A, Rev B, Env A, and Env B, are shown for each animal in SFC per 10⁶ PBMC at Day 74 (14 days prior to infection), Day 28 post-infection (p.i.), and Day 42 p.i. In most instances, the activity decreased significantly by Day 42 p.i. AV89's strong response against Rev (430 SFC/10⁶ PBMC) was not shown, so that the y-axis maximum would be the same for each graph.

Figure 6

Viral sequences from Day 28 are compared to SIVmac239, the challenge virus. Viral RNA was extracted from each animal's plasma on Day 28. After RT-PCR, cDNAs were cloned into a plasmid. Individual clones were then isolated and sequenced. In parentheses, the numerator indicates the number of clones with a given sequence and the denominator shows the total number of clones sequenced. Mutations are highlighted in red.

Figure 7

Encoded amino acids from Day 28 viruses in Tat, Rev, and Env from the overlapping regions used in the peptide vaccination. Proposed CTL epitopes are highlighted in yellow.