Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene

Abstract

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

FIG. 1.

Frequencies of Gag-specific IFN-γ-secreting cells from monkeys immunized with Ad5-gag. These values are expressed as the number of SFC per 10⁶ PBMCs. Naïve monkeys (adenovirus type 5 neutralization (neut) titers < 10) were immunized with various doses of Ad5-gag at weeks 0 and 24. Monkeys that were pretreated with one or three 10¹⁰ viral particle (vp) doses of non-Gag-encoding adenovirus type 5 virus (producing neutralization titers of ≈30 to 90 and ≈270 to 810, respectively, at the time of the first Ad5-gag injection) were immunized with 10¹¹ viral particles of Ad5-gag at weeks 0 and 24. PBMCs collected at weeks 0, 4, 24, and 28 were incubated in the absence (mock) or presence of the HIV-1 Gag peptide pool, and the mock-corrected response levels are shown here for each monkey. Mock-treated background responses averaged 3 SFC per 10⁶ PBMCs (standard deviation, ≈5 SFC per 10⁶ PBMCs).

FIG. 2.

CTL responses in monkeys immunized with Ad5-gag. Peptide-pulsed autologous BLCLs were used as target cells at the indicated effector-to-target cell (E:T) ratios, treated with PBMCs (collected at week 8) from Ad5-gag vaccinees restimulated for 2 weeks with gag-expressing vaccinia virus. The percentages of lysed target cells are shown for unpulsed cells (open squares) and cells pulsed with the HIV-1 Gag peptide pool (solid diamonds).

FIG. 3.

Time course of induction of Gag-specific T-cell responses from PBMCs of representative monkeys that received 10¹¹ viral particles and 10⁹ viral particles of Ad5-gag at week 0 and week 24 (A). Mock responses were subtracted to give the reported levels shown here. Kinetics of induction of p24-specific antibodies as a function of the Ad5-gag dose and adenovirus type 5 preexposure (B). The geometric mean titers (GMT) are shown with standard errors for each cohort of either three or six monkeys. Each curve is defined by the Ad5-gag dose (viral particles, vp) and adenovirus type 5 neutralization titers measured at the start of the immunization. Titers below the dection limit of 10 mMU/ml are scored at 5 mMU/ml (line).

FIG. 4.

Intracellular IFN-γ staining of PBMCs from Ad5-gag vaccinees following overnight incubation in medium alone (mock) or in medium plus the Gag peptide pool (Gag 20-aa). CD3⁺ lymphocytes are shown and were characterized for CD8⁺ staining and IFN-γ production. Numbers reflect the percentage of CD3⁺ lymphocytes that were CD8⁺ IFN-γ⁺ (upper right quadrant) or CD4⁺ IFN-γ⁺ (lower right quadrant).

FIG. 5.

Frequencies of Gag-specific IFN-γ-secreting cells from monkeys immunized with Ad5-gag and MVA-gag. Priming immunizations were administered at weeks 0 and 4, followed by a booster shot at week 24 with the same virus. Shown are the mock-corrected levels against the Gag peptide pool prior to the first dose (pre), 4 weeks after the second dose (post prime), time of the boost (preboost), and 4 weeks after the viral boost (postboost) for each animal.

FIG. 6.

Frequencies of Gag-specific IFN-γ-secreting cells from PBMCs of macaques immunized following various DNA prime-adenovirus type 5 boost regimens. (A) Macaques were given multiple doses of 5 mg of V1Jns-gag DNA intramuscularly in phosphate-buffered saline, with aluminum phosphate (alum) or with CRL1005 (7.5 mg) at weeks 0, 4, and 8. Shown are levels of antigen-specific T cells (mock corrected) at different time points during and after the priming immunizations. (B) Macaques were treated intramuscularly with three doses (weeks 0, 4, and 8) of 5 mg of V1Jns-gag with and without adjuvants (CRL1005 at 7.5 or 22.5 mg) and boosted with an intramuscular dose of Ad5-gag (10⁷ viral particles or 10¹¹ viral particles) at weeks 24 to 26. Shown are the mock-corrected levels prior to the first dose (pre), 4 weeks after the third dose (post prime), time of the boost (preboost), and 4 weeks after the viral boost (postboost) for each animal.

FIG. 7.

Intracellular IFN-γ staining of PBMCs from a representative cohort of macaques immunized with three doses of V1jns-gag formulated with CRL1005-based adjuvant (weeks 0, 4, and 8) followed by 10⁷ viral particles of Ad5-gag boost (week 24). Samples were collected at week 10 (postprime) and week 28 (postboost). Shown are CD3⁺ lymphocytes following incubation with the HIV-1 Gag peptide pool that were characterized for CD8⁺ staining and IFN-γ production. For all samples, intracellular IFN-γ production in the absence of the peptide pool was minimal (data not shown). Numbers reflect the percentage of CD3⁺ lymphocytes that were CD8⁺ IFN-γ⁺ (upper right quadrant) or CD4⁺ IFN-γ⁺ (lower right quadrant) after subtraction of the mock levels.

FIG. 8.

Percentages of Gag-specific T cells that are CD3⁺ CD8⁺ in rhesus macaques immunized with various regimens. The adenovirus type 5 prime-adenovirus type 5 boost (Ad5-Ad5) cohort consisted of animals given two priming doses of the vector (10⁹ or 10¹¹ viral particles) followed by a booster shot of the same dose. The DNA-CRL1005 prime-adenovirus type 5 boost cohort comprised animals given three priming immunizations with 5 mg of V1Jns-gag formulated with CRL1005-based adjuvant, followed by 10⁷ viral particles of Ad5-gag. The percentage values are reported for this cohort of 11 animals before (DNA-CRL1005) and after the boost (DNA-CRL1005-Ad5). The dark diamonds and bars are the cohort arithmetic means and standard errors of the mean, respectively. These data points include those for the animals in Fig. 4 and 7.