Non-immunogenicity of overlapping gag peptides pulsed on autologous cells after vaccination of HIV infected individuals

Abstract

Background: HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.

Methodology: We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach 'OPAL-HIV-Gag(c)'. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).

Results: The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).

Conclusion/significance: Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.

Name of registry: ClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search.

Figure 1. Study subject disposition and allocation to dosing cohorts.

(A): Thirty eight subjects were screened for the study, with 23 randomised and 18 completing the study. (B): Diagram showing the planned study allocation to dose escalating cohorts (5∶2) and with sentinel cohorts (1∶1) shown for the 12 mg, 24 mg and 48 mg dose groups.

Figure 2. Magnitude and expansion potential of pre-existing OPAL-HIV-Gag(c) specific responses.

Eighteen subjects completing the study were tested for IFNγ ELIspot responses expressed as SFU per million inpuT-cells to OPAL-HIV-Gag(c) peptides or mock (media only) from fresh ex vivo PBMCs (A) or from 10 day cultured OPAL-HIV-Gag(c) peptide expanded PBMCs (B) from screening samples available at 2–6 weeks prior to baseline. The expansion capacity was determined as the fold change of magnitude for the cultured ELIspot over the ex vivo ELIspot (C). ND not done.

Figure 3. OPAL-HIV-Gag(c) peptide pool specific responses before and after vaccination.

All six subjects from each dose group (0 mg, 12 mg and 24 mg) were tested for OPAL-HIV-Gag(c) specific or no peptide (mock) responses by IFNγ ex vivo ELIspot performed from fresh cells at week 0, 10, 12, 13, 14 and 16 after first vaccination expressed as the mean SFU per million cells of triplicate stimulations (A) and expressed as median values within dose groups with error bars representing inter quartile ranges (B).

Figure 4. OPAL-HIV-Gag(c) and CMV peptide pool specific CD8+ T-cell responses before and after vaccination.

All six subjects from each dose group (0 mg, 12 mg and 24 mg) were tested for OPAL-HIV-Gag(c) specific or no peptide (no stimulation) responses by ICS shown as IFNγ+/MIP1β+ double positive CD8+ T-cells processed from frozen PBMCs derived at week 0, 13 and 14 after first vaccination expressed as the mean of triplicate stimulations (A) and expressed as median values within dose groups with error bars representing inter quartile ranges for OPAL-HIV-Gag(c) (B) and for CMV specific CD8+ T-cell responses (C).

Figure 5. OPAL-HIV-Gag(c) and CMV peptide pool specific CD4+ T-cell responses before and after vaccination.

All six subjects from each dose group (0 mg, 12 mg and 24 mg) were tested for OPAL-HIV-Gag(c) specific or no peptide (mock) responses by ICS shown as IFNγ+/MIP1β+ double positive CD4+ T-cells processed from frozen PBMCs derived at week 0, 13 and 14 after first vaccination expressed as the mean of triplicate stimulations (A) and expressed as median values within dose groups with error bars representing inter quartile ranges for OPAL-HIV-Gag(c) (B) and for CMV specific CD4+ T-cell responses (C).

Figure 6. Transient and treatment specific lymphopenia after vaccination.

Total lymphocyte counts were performed before, during and at follow up after vaccination as indicated on the x-axis for the three groups (0 mg, 12 mg and 24 mg) and shown as mean values (million lymphocytes per ml whole blood) for the 6 subjects within each group with normal high and low values for HIV positive individuals indicated by dotted lines (A). Arrows indicate vaccinations. The percent (%) change of lymphocyte count from baseline (week 0) is shown as mean values for the three dose groups with error bars representing standard error of mean (SEM) (B). Only one subject (024) was available for the 48 mg dose group at week 0 and 4.