Enhancing efficacy of HIV gag DNA vaccine by local delivery of GM-CSF in murine and macaque models

Abstract

Controlled release of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific cytotoxic T lymphocyte (CTL) and T helper (Th) responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific antibody (Ab), Th, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and Th, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design.

FIG. 1

(A) Electron and (B) optical micrographs of albumin-heparin microparticles. (C) Release kinetics of MuGM-CSF from mouse serum albumin-heparin Mps in MEM with 10% FBS (MEM 10). Albumin-heparin Mps containing GM-CSF protein were prepared and incubated in MEM 10. The supernatant was refreshed every day, and the bioactive concentration of GM-CSF was measured using a TF-1 cell proliferation assay.

FIG. 2

HIV Gag-specific Th response (A) and CTL-specific lysis (B) to pGAGINS1 DNA vaccine. BALB/c mice (n = 5) were immunized intramuscularly with 20 μg DNA vaccine with bolus GM-CSF, GM-CSF Mps, or blank Mps at weeks 0 and 2. DNA vaccine alone was used as control. Animals were studied 2 weeks after the second immunization.

FIG. 3

Gag-specific Th response (A) and CTL-specific lysis (B) induced by 20 μg Gag DNA vaccine with or without Mps encapsulating 1 μg GM-CSF or 10 μg or 20 μg of GM-CSF plasmids. BALB/c mice (n = 5) were inoculated intramuscularly at weeks 0 and 2.

FIG. 4

Gag-specific CTL response in individual BALB/c mice immunized with HIV plasmid and GM-CSF Mps. Animals were immunized twice with 20 μg Gag DNA vaccine with Mps encapsulating 1 μg GM-CSF twice at a 2-week interval.

FIG. 5

Effect of 3 mg Gag encoding plasmid immunization in the presence of Mps encapsulating 20 μg HuGM-CSF and 6 μg HuTNF-a on immune response in individual rhesus monkeys. (Top) Basal responses (week 0) of Gag-specific CTL, Th, and Ab. (Bottom) Immune responses 4 weeks postimmunization.

FIG. 6

Gag-specific immune response induced by DNA priming and DNA boosting with 3 mg Gag encoding plasmids in the presence of Mps encapsulating 20 μg HuGM-CSF and 6 μg HuTNF-α in individual rhesus monkeys at weeks 8, 12, and 16. After the first immunization, the 4 rhesus monkeys were vaccinated three more times at monthly intervals using the strategy described in Materials and Methods. PBMCs and sera were collected 1 month after each inoculation and assayed.

FIG. 7

Boosting with Gag p55 protein and CFA (top) or Gag p55 protein and cytokine Mps (bottom). Gag-specific CTL, Th, and Ab responses 4 weeks after rhesus monkeys were immunized with 200 μg HIV Gag p55 protein, mixed with CFA (1:1) or 200 μg Gag p55 protein-encapsulated Mps and 20 μg GM-CSF/6 μg TNF-α Mps.

SCHEME 1

Immunization protocol of rhesus monkeys.