Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine

Abstract

Background: Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine.

Methods: VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by enzyme-linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme-linked immunospot assay and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured.

Results: The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4(+) and CD8(+) T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of interferon- gamma -producing T cells and an increased frequency of interleukin-2-producing T cells at 1 year.

Conclusions: DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.

Trial registration: ClinicalTrials.gov NCT00047931.

Figure 1

Schematic of the DNA vaccine design. Four separate DNA plasmids were produced by inserting individual HIV-1 gene constructs into the pVR1012 backbone under the control of the cytomegalovirus (CMV) immediate-early (IE) promoter, followed by the bovine growth hormone polyadenylation (bGH poly A) sequence [3, 12]. The synthetic gag gene is from the clade B strain HXB2 (GenBank accession no. K03455), the synthetic pol gene (pol/h) is from the clade B strain NL4-3 (GenBank accession no. M19921), and the synthetic nef gene (nef/h) is from the clade B strain PV22 (GenBank accession no. M19921). Mutations (indicated by Xs), including the deletion of the carboxy-terminus of Gag (indicated by the triangle), were introduced in the protease and reverse-transcriptase genes to prevent processing of the pol gene products and to reduce the potential for functional enzymatic activity. This resulted in a fusion protein that directly reads through the frame shift in Gag (F2) through Pol and into Nef. This gene product is not able to assemble or produce pseudoparticles. To create synthetic gp145, versions of the envelope genes were truncated immediately downstream of the transmembrane domain of gp41. In each construct, the cleavage site and fusion peptide at the junction of gp120 and gp41 were deleted, and a portion of the interspace between the 2 heptad-repeat regions in gp41 was deleted. The Env gene products are primarily cell associated rather than secreted. The EnvA sequence is from 92rw020 (CCR5 tropic; GenBank accession no. U08794), the EnvB sequence is from HXB2 (CXCR4 tropic; GenBank accession no. K03455), and the EnvC sequence is from 97ZA012 (CCR5 tropic; GenBank accession no. AF286227). HR1–HR2, heptad-repeat regions in gp41; IN, integrase; NC, nucleocapsid; PR, protease; V1–V5, variable regions in envelope.

Figure 2

Induction, specificity, and dose response of the vaccine-induced antibody response to clades A, B, and C Env proteins. A, Western blot analysis of Env antigens captured by immunoprecipitation using prevaccination (pre) and 12-week (post) serum samples from representative placebo recipients (0 mg) and vaccine recipients (4- or 8-mg doses). Arrows indicate the EnvB-specific band distinguished from a small nonspecific (NS) background band. B, Specific reactivity of antibody induced by vaccination to EnvA, EnvB, and EnvC but not to an unrelated Ebola virus glycoprotein–negative control (Ebola GP). All proteins were produced in the supernatants of 293T cells transfected with the vaccine plasmids. Arrows indicate the Env-specific band. Results are shown for representative subjects from the placebo group (0 mg) and from the 4- and 8-mg groups. No positive bands were detected for any of the placebo recipients at any time point in the study. C, Frequency of positive antibody responders to purified proteins at week 12 as measured by end-point titration ELISA (hatched bars) or by immunoprecipitation followed by Western blotting (black bars), as a function of dose. No antibody was detected by these methods for placebo recipients or for vaccine recipients before immunization. D, Peak response to purified proteins for EnvA, EnvB, and EnvC as measured by end-point titration ELISA for each subject, by dose group. The peak response (reciprocal titer) occurred between weeks 8 and 24. The box plots indicate the median, 25th, and 75th percentiles for each dose level, and the error bars show the 5th and 95th percentile.

Figure 3

Measurement of HIV-specific T cell responses. The T cell response measurements were largely based on flow-cytometric detection of intracellular cytokine production in peripheral-blood mononuclear cells (PBMCs) stimulated with peptide pools representing the vaccine antigen. Because this is a relatively new approach for determining immunogenicity in clinical trials of preventive vaccines, an example of the primary data and gating strategy for this method is demonstrated for a representative subject. CD4⁺ and CD8⁺ T cell responses are shown for a single recipient of the 4-mg dose at week 12 (4 weeks after completion of the vaccination schedule). PBMCs were incubated either with costimulatory antibodies only (negative control) or with EnvA, EnvB, or EnvC peptide pools, and production of interferon (IFN)–γ and/or interleukin (IL)–2 was measured on the same wavelength. Gating (pink box) reflects cells producing higher levels of cytokine. The numeric value above each gate represents the percentage of total CD4⁺ or CD8⁺ T cells producing cytokine.

Figure 4

Magnitude of T cell responses to specific vaccine components. T cell responses were measured by intracellular cytokine staining (ICS) assay to detect interferon (IFN)–γ and/or interleukin-2 and by IFN-γ enzyme-linked immunospot (ELISpot) assay for all placebo and vaccine recipients. A, Median magnitudes of peptide pool–specific responses, shown as a percentage of total CD4⁺ or CD8⁺ T cells for the ICS assay (scale 0–0.1) and as the no. of spot-forming cells per 10⁶ peripheral-blood mononuclear cells (PBMCs) for the ELISpot assay (scale 0–150), for all subjects, by dose group. The time course for each subject is shown by study week on the X-axis; arrows along the top of the graph designate time points for immunizations (study weeks 0, 4, and 8). Within each of the 24 boxes, the entire time course of the study is represented. Each column shows the responses to a peptide pool representing the respective vaccine antigens (EnvA, EnvB, EnvC, Gag, Nef, and Pol). Each row represents a dose cohort (placebo recipients, 2-mg recipients, 4-mg recipients, and 8-mg recipients). Red bars represent CD4⁺ T cell responses as measured by ICS assay, green bars represent CD8⁺ T cell responses as measured by ICS assay, and blue bars represent CD4⁺ or CD8⁺ T cell responses as measured by ELISpot assay. Time points without samples for analysis are represented by asterisks. B, Magnitudes of specific T cell responses to a peptide pool for EnvA at study week 12, by dose group. EnvA-specific responses are shown for each subject as measured by 3 assays; shown are the percentages of CD4⁺ or CD8⁺ T cells producing cytokine as measured by ICS assay and the no. of spot-forming cells per 10⁶ PBMCs as measured by ELISpot assay. The left plot shows EnvA-specific CD4⁺ T cell responses, the middle plot shows EnvA-specific CD8⁺ T cell responses, and the right plot shows EnvA-specific CD4⁺ or CD8⁺ responses. The box plots indicate the median, 25th, and 75th percentiles for each dose level, and the error bars show the 5th and 95th percentiles. The horizontal dashed line on each plot indicates the laboratory threshold of positivity for each assay (for the ICS assay, 0.0241% for CD4⁺ T cells and 0.0445% for CD8⁺ T cells; for the ELISpot assay, 50 sfc/10⁶ PBMCs).

Figure 5

Frequencies of subjects with detectable T cell responses. All T cell responses for each antigen are shown for all subjects at all time points. Each of the 24 boxes shows the frequency of response to a different antigen in a different dose group for the entire time course of the study. The Y-axis of each of the 24 boxes shows the frequency of response as the percentage of subjects in a dose group with a positive response to the respective peptide pool for each assay. The X-axis shows the time course of the study by study week (0–52) in each of the 24 plots. Each row represents a dose cohort (placebo recipients, 2-mg recipients, 4-mg recipients, and 8-mg recipients). Each column represents the peptide pool used for stimulation of peripheral-blood mononuclear cells (EnvA, EnvB, EnvC, Gag, Nef, and Pol). Red bars represent CD4⁺ T cell responses as measured by intracellular cytokine staining (ICS) assay, green bars represent CD8⁺ T cell responses as measured by ICS assay, and blue bars represent CD4⁺ or CD8⁺ T cell responses as measured by enzyme-linked immunospot (ELISpot) assay. No week 2 data and only 1 sample from week 6 are included for the 2-mg cohort (time points without samples for analysis are represented by asterisks).

Figure 6

Evolution of cytokine-expression patterns in vaccine-induced T cells over time. Multiparameter flow cytometry was performed in a subset of subjects to define the relative frequency of interferon (IFN)–γ and interleukin (IL)–2 production in T cells stimulated by the vaccine-specific peptide pools. A, Response of either CD4⁺ or CD8⁺ T cells from a representative vaccine recipient is shown at study week 12 after stimulation with the EnvA peptide pool. For each of the 2 plots, the Y-axis shows CD3⁺ (CD4⁺ or CD8⁺) T cells producing IFN-γ, whereas the X-axis shows IL-2 production. The plot on the left demonstrates CD4⁺ T cell production of IL-2 alone (in the bottom right quadrant; 0.096%), IFN-γ alone (in the top left quadrant; 0.028%), or both (in the top right quadrant; 0.030%). The plot on the right demonstrates CD8⁺ T cell production of IFN-γ alone (in the top left quadrant; 0.52%), IL-2 alone (in the bottom right quadrant; 0.075%), or both (in the top right quadrant; 0.003%). Over time, the relative frequency of cells producing IFN-γ appeared to diminish, and the relative frequency of vaccine-specific IL-2–producing cells appeared to increase, as shown in panel B by use of pie charts demonstrating the average cytokine-expression pattern from a subset of vaccine recipients with the highest initial responses to the EnvA peptide pool over the 52-week time course of the study. There are 2 sets of pie charts: the top row shows CD4⁺ T cells, and the bottom row shows CD8⁺ T cells, both as measured by intracellular cytokine staining assay. Five time points of the study are represented (week 10, 12, 24, 38, and 52), and the no. of representative subjects included at each time point is listed. The pie charts show the distribution of cytokine-expression patterns of each T cell responding to the EnvA peptide pool: the blue segments represent T cells producing IL-2 only, the red segments represent T cells producing both IL-2 and IFN-γ, and the green segments represent T cells producing IFN-γ only.