Sustained peptide-specific gamma interferon T-cell response in rhesus macaques immunized with human immunodeficiency virus gag DNA vaccines

Abstract

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV gag gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8(+) T cells. Finally, we examined the fine specificity of the T-cell response to the gag vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.

FIG. 1.

IFN-γ ELISPOT response of rhesus monkeys to an HIV gag DNA vaccine formulated with CRL8623 adjuvant. Groups of three monkeys were injected i.m. with 5 mg of HIV gag DNA formulated with CRL8623 (circles) or with saline (squares) at 0, 4, and 8 weeks. ELISPOT responses were enumerated at the indicated times postvaccination, using PBMCs that were unfractionated (closed symbols) or depleted of CD4 T cells (open symbols).

FIG. 2.

Response profiles representative of IFN-γ ELISPOT responses of rhesus monkeys to HIV gag DNA vaccines. Rhesus monkeys were injected i.m. with 5 mg of HIV gag plasmid DNA vaccine at weeks 0, 4, 8, and 24. PBMCs collected at week 51 were tested for their response to individual 20-mer peptides comprising the HIV Gag sequence. Unseparated (unsep) and CD4-depleted (CD−) PBMCs were tested in parallel for reactivity to each of 50 peptides. (a) Example of a CD4-dominated response in which four distinct peptides were recognized. (b) Results for a monkey that responded to a single peptide. Note that the magnitude of the response increased following CD4 depletion, indicating that the response was predominantly CD8 mediated. (c) Example of a mixed-phenotype response yielding a strong CD8 response to one peptide and an equally vigorous CD4 response directed to two distinct peptides.

FIG. 3.

Profile of ELISPOT responses to individual 20-mer peptides from HIV Gag. PBMCs from 23 monkeys immunized with three or four doses of HIV gag DNA vaccine were cultured at a density of 2 × 10⁵ cells per well with each of 50 overlapping 20-mer peptides (offset by 10 amino acids). Results are expressed as the total IFN-γ ELISPOT response (adjusted per million PBMCs) to each peptide for all 23 monkeys. The number in parentheses above each column indicates the number of monkeys that responded to the corresponding peptide at a level of ≥50 SFC per 10⁶ PBMCs.