Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector

Abstract

Background: The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a replication-defective recombinant adenovirus serotype 5 (rAd5) vector HIV-1 candidate vaccine.

Methods: The vaccine is a mixture of 4 rAd5 vectors that express HIV-1 subtype B Gag-Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n=6) or vaccine at dose levels of 10(9) (n=10), 10(10) (n=10), or 10(11) (n=10) particle units and were followed for 24 weeks to assess immunogenicity and safety.

Results: The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen-specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4(+) and CD8(+) T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme-linked immunospot assay. Env-specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme-linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected.

Conclusions: A single injection induced HIV-1 antigen-specific CD4(+) T cell, CD8(+) T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV-1 vaccine is now being evaluated in combination with a multiclade HIV-1 DNA plasmid vaccine.

Trial registration: ClinicalTrials.gov NCT00083330.

Figure 1

Schematic of the design of the replication-defective recombinant adenovirus serotype 5 (rAd5) vector vaccine. Four separate rAd5 vectors were produced using the same genetic backbone and manufacturing approaches. The HIV-1 vaccine antigen–expression cassettes in the E1 region contained the immediate-early cytomegalovirus (CMV) enhancer/promoter (GenBank accession no. X17403; nucleotide positions 174314−173566), positioned right to left with respect to the viral E1 region. This was followed by an artificial untranslated region of 144 bp and 3′ splice-site sequences, the open reading frame (ORF) of the gene to be expressed, and the simian virus 40 polyadenylation signal (SV40 poly A). The E4 region and a portion of the E3 region were also deleted as shown. A fusion Gag/Pol polyprotein was encoded by a synthetic ORF with nucleotide sequences based on the gag gene from clade B strain HXB2 (GenBank accession no. K03455) and the pol gene (pol/h) from clade B strain NL4−3 (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, reducing the potential for functional enzymatic activity [12]. This resulted in a fusion protein that directly reads through the frame shift in Gag (F2). To create synthetic gp140 versions of the Env genes truncated at the transmembrane domain of gp41, sequences from clade A strain 92rw020 (CCR5 tropic; GenBank accession no. U08794), clade B strain HXB2 (X4 tropic; GenBank accession no. K03455) with V1 and V2 deleted and V3 replaced with BaL sequence (GenBank accession no. M68893), and clade C strain 97ZA012 (CCR5 tropic; GenBank accession no. AF286227) were used [13]. In each construct, the cleavage site and fusion peptide at the junction of gp120 and gp41 was deleted, and a portion of the interspace between the 2 heptad-repeat regions in gp41 was deleted. In addition, deletion of the V1/V2 loops from the EnvB construct was required to improve the stability of the vector during manufacturing. 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 vaccine-induced antibody to clades A, B, and C Env proteins. A, Western blot analysis of the antigen captured by immunoprecipitation using prevaccination (pre) and week 4 (post) serum samples from 3 representative subjects in the placebo group and in the 3 dose groups receiving 10⁹, 10¹⁰, or 10¹¹ particle units (PUs). The analysis shows that Env-specific antibody is induced by the vaccine; arrows indicate the EnvB-specific band. B, Recognition of vaccine-induced antibody. Serum from 1 subject representing each of the dose groups shows that vaccine-induced antibody recognized all 3 Env subtypes but not the Ebola virus glycoprotein–negative control (Ebola GP). Arrows indicate the Envspecific band. No positive bands were detected in any of the placebo recipients at any time point in the study. C, Frequency of positive antibody responders to EnvB at week 4 as measured by immunoprecipitation followed by Western blotting, for each dose group. D, Geometric means of the reciprocal dilution of antibody to purified gp140 for EnvA, EnvB, and EnvC for each subject at week 4, by dose group. Titers were determined by endpoint titration ELISA. Error bars represent SDs. The dilution series began at 1:30, and negative samples were assigned a value of 1:15. The proteins used for ELISA were between 85% and 90% pure as determined by Western blotting and polyacrylamide gel electrophoresis.

Figure 3

Frequency of subjects with detectable T cell responses. T cell responses to each peptide pool in all dose groups are shown. Each box shows the entire time course for each T cell assay. The Y-axis of each box shows the frequency of positive responders to the respective peptide pool for each assay as percentage of subjects in a dose group (0%−100%). Red bars indicate CD4⁺ T cell responses as measured by intracellular cytokine staining (ICS) assay, green bars indicate CD8⁺ T cell responses as measured by ICS assay, and blue bars indicate CD4⁺ or CD8⁺ T cell responses as measured by enzyme-linked immunospot assay (ELISpot).

Figure 4

Magnitude of T cell responses to specific vaccine components. T cell responses to each peptide pool for each dose group 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), by dose group. Both values are plotted on a linear scale. Each box shows the 24-week time course of the study for each T cell assay. Red bars indicate CD4⁺ T cell responses as measured by ICS assay, green bars indicate CD8⁺ T cell responses as measured by ICS assay, and blue bars indicate CD4⁺ or CD8⁺ T cell responses as measured by ELISpot assay. B, Magnitudes of EnvA-specific T cell responses at study week 4 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, by dose group. The box plots indicate the median, 25th, and 75th percentiles for each dose level, and the error bars show the 5th and 95th percentile. The horizontal dashed line on each plot indicates the threshold of positivity.

Figure 5

Combined cellular responses to vaccine antigens. The highest Env response to a single subtype was added to the Gag and Pol responses for each subject as a measure of the total vaccine-induced T cell response, and the median for each dose group was plotted on a linear scale for the intracellular cytokine staining (ICS) and the enzyme-linked immunospot (ELISpot) assays. A, Median magnitudes for maximum Env (blue), Gag (grey), and Pol (brown) are shown as a percentage of total CD4⁺ or CD8⁺ T cells producing cytokine for the ICS assay and as the no. of spot-forming cells per 10⁶ peripheral-blood mononuclear cells (PBMCs) for the ELISpot assay. Each box represents the 24-week time course; the scale is 0%−0.2% of the total T cell subset for the ICS data and 0−300 sfc/10⁶ PBMCs for the ELISpot data. B, Median magnitudes of total T cell response at week 4 as measured by the ICS assay (percentage of total CD4⁺ or CD8⁺ T cells producing cytokine) and the ELISpot assay (no. of spot-forming cells per 10⁶ PBMCs). Subjects are grouped by preimmunization 90% adenovirus serotype 5 (Ad5) neutralization titers (<1:12 or ≥1:12); at screening, the proportion of subjects with an Ad5 antibody titer <1:12 was 6 (100%) of 6 in the placebo group, 3 (30%) of 10 in the group receiving 10⁹ particle units (PUs), 4 (40%) of 10 in the 10¹⁰-PU group, and 7 (70%) of 10 in the 10¹¹-PU group. The scale is 0%−0.15% of the total T cell subset for the ICS data and 0−300 sfc/10⁶ PBMCs for the ELISpot data.