Multifunctional T-cell characteristics induced by a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine regimen given to healthy adults are dependent on the route and dose of administration

Abstract

A phase I clinical vaccine study of a human immunodeficiency virus type 1 (HIV-1) vaccine regimen comprising a DNA prime formulation (5-valent env and monovalent gag) followed by a 5-valent Env protein boost for seronegative adults was previously shown to induce HIV-1-specific T cells and anti-Env antibodies capable of neutralizing cross-clade viral isolates. In light of these initial findings, we sought to more fully characterize the HIV-1-specific T cells by using polychromatic flow cytometry. Three groups of participants were vaccinated three times with 1.2 mg of DNA administered intradermally (i.d.; group A), 1.2 mg of DNA administered intramuscularly (i.m.; group B), or 7.2 mg of DNA administered i.m. (high-dose group C) each time. Each group subsequently received one or two doses of 0.375 mg each of the gp120 protein boost vaccine (i.m.). Env-specific CD4 T-cell responses were seen in the majority of participants; however, the kinetics of responses differed depending on the route of DNA administration. The high i.m. dose induced the responses of the greatest magnitude after the DNA vaccinations, while the i.d. group exhibited the responses of the least magnitude. Nevertheless, after the second protein boost, the magnitude of CD4 T-cell responses in the i.d. group was indistinguishable from those in the other two groups. After the DNA vaccinations and the first protein boost, a greater number of polyfunctional Env-specific CD4 T cells (those with > or = 2 functions) were seen in the high-dose group than in the other groups. Gag-specific CD4 T cells and Env-specific CD8 T cells were seen only in the high-dose group. These findings demonstrate that the route and dose of DNA vaccines significantly impact the quality of immune responses, yielding important information for future vaccine design.

FIG. 1.

Induction of Env-specific CD4 and CD8 T-cell responses. Representative intracellular cytokine staining data show the production of cytokines IFN-γ (A) and IL-2 (B), the upregulation of CD154 by CD4 T cells (C), and the production of IFN-γ by CD8 (D) and CD8⁺ CD107⁺ (E) T cells. Data represent Env-specific responses in a group C vaccinee at baseline, day 98 (DNA), and day 154 (Prot-1).

FIG. 2.

Frequency of CD4 T-cell responses in vaccinees. The percentages of subjects in each of the three groups, group A (given a low dose i.d.), group B (given a low dose i.m.), and group C (given a high dose i.m.), that were positive for IFN-γ at various time points postvaccination are shown. Env-A- and Env-B-specific responses were significantly more frequent in group C than in the other groups (*, P ≤ 0.001, and **, P ≤ 0.05, respectively; Fisher's exact test). DNA, day 98.

FIG. 3.

Comparison of the magnitudes of the Env-specific CD4⁺ T-cell responses. The production of IFN-γ, IL-2, and TNF-α and the upregulation of CD154 in each of the three groups, i.e., groups A, B, and C, at 2 weeks after DNA administration and 2 weeks after each of the two protein booster doses (Prot-1 and Prot-2) were analyzed. Statistical comparisons were made using the Mann-Whitney U test.

FIG. 4.

Magnitudes of CD154⁺ CD4⁺ cytokine-producing T cells. The production of IFN-γ, IL-2, or TNF-α by CD154⁺ CD4⁺ T cells in groups A, B, and C after stimulation with Env-A, Env-B, and Gag antigens was measured. Data represent the median responses with interquartile ranges. For each antigen, data are shown for the baseline, 2 weeks after DNA administration, 2 weeks after each of the protein booster doses (Prot-1 and Prot-2), and closeout as five vertical bars on the x axis. The horizontal bars on the x axis represent all five time points for each antigen. Responses were significant (P < 0.05; Wilcoxon text) in all instances in which the interquartile range at baseline did not overlap with the median for the DNA, Prot-1, Prot-2, or study closeout time point.

FIG. 5.

Induction of polyfunctional CD4 T cells. Pie charts demonstrate the functions of the CD4 T cells (IFN-γ, IL-2, and/or TNF-α production and/or CD154 upregulation) 2 weeks after DNA administration (DNA) and 2 weeks after each of the two protein vaccinations (Prot-1 and Prot-2) for group A (a), group B (b), and group C (c). Underneath each set of pie charts, bar graphs show all possible combinations of the one to four functions on the x axis, with the bold line along the x axis covering all the possible subcombinations for a given function. All subcombinations for a given function are identified by a single color that is also represented as a slice in the pie chart. For each of the 15 functions shown, a bar represents the median response expressed as a percentage of the total CD4 T-cell response (y axis) at post-DNA administration (DNA; blue), Prot-1 (red), and Prot-2 (green) time points. For group C, data for time points following the administration of the DNA vaccine and the first protein booster dose only are shown, as this group received a single protein booster dose. c, CD154; g, IFN-γ; i, IL-2; t, TNF-α.

FIG. 6.

Surrogate markers of memory phenotypes on Env-specific CD4⁺ T cells. Data shown are representative of the phenotypic characterization of antigen-specific CD4 T cells after the second protein boost in a volunteer from group B (given a low dose i.m.). Surface markers for memory on Env-specific CD4⁺ T cells in select combinations (CD127, CCR7 versus CD45RA, CD27 versus CD45RO, and CD27 versus CD43) are shown. The production of IL-2 (red dots) over that of the base population (gray) in response to Env antigen and staphylococcal enterotoxin B (SEB), along with data for the unstimulated sample (control), is shown. Pseudocolor plots of the select combinations are shown in the far-right column.