Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys

Abstract

The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However, recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys, divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant and sustained in vivo activation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited humoral and cellular immune responses in higher primates.

Figure 1

Vaccine trial design. Sixteen monkeys were divided into four experimental groups and were immunized with: 1) 10 mg of sham plasmid; 2) 5 mg of HIV-1 Env 89.6P and 5 mg of SIV mac239 Gag DNA vaccines; 3) DNA vaccines and IL-2/Ig protein; or 4) DNA vaccines and IL-2/Ig plasmid. Five milligrams of sham plasmid was administered to the animals that received the DNA vaccines alone on day 2 after the week 0 and week 4 immunizations. IL-2/Ig protein (0.5 mg/day) was administered by twice-daily i.m. injections on days 1–14 after the week 0 and week 4 immunizations. Five milligrams of IL-2/Ig plasmid was administered on day 2 after the week 0 and week 4 immunizations. The week 8 immunization was performed without cytokine administration.

Figure 2

IL-2/Ig protein and IL-2/Ig plasmid administration lead to significant CD25 expression on CD3+ PBL of the vaccinated monkeys. The monkeys were bled at week 0, week 1, week 2, and every 2 weeks thereafter. Gated CD3+ lymphocytes from whole blood were examined for staining with an anti-CD25 mAb by flow cytometry. The monkeys that received IL-2/Ig protein demonstrated a striking rise and rapid fall of CD25 expression. The monkeys that received IL-2/Ig plasmid showed a less dramatic but more sustained increase in CD25 expression. Means and SE for each group are shown.

Figure 3

IL-2/Ig protein and IL-2/Ig plasmid administration augment vaccine-elicited anti-Env antibody responses in monkeys. Plasma was obtained from the vaccinated animals every 2 weeks and analyzed for anti-Env antibodies by ELISA. Sham-vaccinated monkeys did not have detectable anti-Env antibody titers. The most striking and consistent augmentation of anti-Env antibody titers was observed in the animals that received IL-2/Ig plasmid. Geometric mean titers and SE for each group are shown.

Figure 4

Evolution of the SIV Gag p11C-specific CTL response detected by tetramer staining and functional cytotoxicity assays in a representative vaccinated monkey. Tetramer staining of fresh PBL, tetramer staining of p11C-stimulated PBL, and functional lysis of p11C-pulsed targets by epitope peptide-stimulated PBL are shown for the following time points: week 0 (preimmune), week 2 (2 weeks after the primary immunization), week 6 (2 weeks after the second immunization), and week 10 (2 weeks after the third immunization). Staining with the Mamu-A*01/p11C tetramer was assessed by four-color flow cytometry on gated CD3+CD8+ lymphocytes. Functional lysis was determined by using p11C epitope peptide- and p11B control peptide-pulsed target cells. Tetramer staining was evident in peptide-stimulated PBL after the primary immunization and in fresh PBL after the second immunization. Functional lysis correlated well with tetramer staining. This particular monkey (772) received the DNA vaccines and IL-2/Ig protein.

Figure 5

IL-2/Ig protein and IL-2/Ig plasmid administration augment SIV Gag p11C-specific CTL responses in monkeys after primary immunization. p11C-specific CTL responses were analyzed every 2 weeks by tetramer staining (A) and functional cytotoxicity assays (B) using peptide-stimulated PBL. Staining with the Mamu-A*01/p11C tetramer was assessed by four-color flow cytometry on gated CD3+CD8+ lymphocytes. Specific functional lysis was determined by using chromium-release cytotoxicity assays at effector-to-target ratios of 10:1. The most consistent and significant augmentation was observed in the monkeys that received IL-2/Ig plasmid. Means and SE for each group are shown.

Figure 6

IL-2/Ig protein and IL-2/Ig plasmid administration augment SIV Gag p11C-specific CTL responses in fresh PBL from the vaccinated monkeys. p11C-specific CTL responses were analyzed every 2 weeks by tetramer staining of fresh PBL. Staining with the Mamu-A*01/p11C tetramer was assessed by four-color flow cytometry on gated CD3+CD8+ lymphocytes. The most consistent and significant augmentation was observed in the monkeys that received IL-2/Ig plasmid. Means and SE for each group are shown.