Clinical evaluation of safety and immunogenicity of PADRE-cytomegalovirus (CMV) and tetanus-CMV fusion peptide vaccines with or without PF03512676 adjuvant

Abstract

Background: It has been reported that cytomegalovirus (CMV) pp65-specific T cells can protect hematopoietic cell transplant (HCT) recipients from CMV complications. Two candidate CMV peptide vaccines composed of the HLA A*0201 pp65(495-503) cytotoxic CD8(+) T-cell epitope fused to 2 different universal T-helper epitopes (either the synthetic Pan DR epitope [PADRE] or a natural Tetanus sequence) were clinically evaluated for safety and ability to elicit pp65 T cells in HLA A*0201 healthy volunteers.

Methods: Escalating doses (0.5, 2.5, 10 mg) of PADRE or Tetanus pp65(495-503) vaccines with (30 adults) or without (28 adults) PF03512676 adjuvant were administered by subcutaneous injection every 3 weeks for a total of 4 injections.

Results: No serious adverse events were reported, although vaccines used in combination with PF03512676 had enhanced reactogenicity. Ex vivo responses were detected by flow cytometry exclusively in volunteers who received the vaccine coadministered with PF03512676. In addition, using a sensitive in vitro stimulation system, vaccine-elicited pp65(495-503) T cells were expanded in 30% of volunteers injected solely with the CMV peptides and in all tested subjects receiving the vaccines coinjected with PF03512676.

Conclusions: Acceptable safety profiles and vaccine-driven expansion of pp65(495-503) T cells in healthy adults support further evaluation of CMV peptide vaccines combined with PF03512676 in the HCT setting.

Clinical trials registration: NCT00722839.

Figure 1.

Peptide vaccines and vaccination schedule. A, Details of the primary structure of the peptide immunogens are shown for PADRE-cytomegalovirus (CMV) (top row) and Tet-CMV (bottom row) fusion peptide vaccines. The synthetic peptides were manufactured using good manufacturing practices (GMP)–grade material; the final products contained l-amino acids. X* indicates cyclohexylalanine. A GMP-grade solution of 10 mM sodium acetate, pH 4.2, was used as a diluent for both peptides. CTL, cytotoxic T lymphocyte. B, Potency tests in mice of the GMP-grade peptide vaccines to confirm their biologic activity in HHDII transgenic mice, immunized with 100 nmol of each CMV peptide vaccine with or without PF03512676. The plots show percentages of cytotoxicity at different effector-to-target ratios (E:T) detected in a 4-h chromium release assay against either relevant (pp65_495-503, solid circles) or control (p53_149-157, open circles) target cells [28]. Upper panels show results from mice immunized with Tet-CMV (left) and Tet-CMV + PF03512676 (right). Lower panels show results from mice immunized with PADRE-CMV (left) and PADRE-CMV + PF03512676 (right). Data in each plot are representative of at least 6 immunized mice. C, Each vaccine (VAC, either PADRE-CMV or Tet-CMV vaccines ± adjuvant) was administered subcutaneously in 4 injections at each dose level (1–3 as shown) for both trial A (CMV peptide vaccines without adjuvant) and trial B (CMV peptide vaccines with adjuvant). Days of vaccination (x-axis) are indicated by the arrows. The type of CMV peptide vaccines used at each dose level is shown. *Timing of blood draws for lab evaluations and immune assessments. D, Eligible subjects were assigned to trial A or trial B based on the time of their enrollment. Trial A dose level (DL) 1 started the study and was staggered such that Tet-CMV subjects were treated prior to PADRE-CMV subjects (solid arrows). At the completion of a DL in trial A, there was progression to the DL + 1 in trial A (dotted arrows) and to the DL in trial B. As mandated by the US Food and Drug Administration, no dose level began in trial B until after that level was safely completed in trial A.

Figure 2.

Tetramer binding before and after vaccination. A, Levels of pp65_495-503 tetramer binding (expressed in percentages) among cytomegalovirus (CMV)–seropositive (solid circles) and CMV-seronegative (open circles) volunteers (x-axes) before vaccination. The bars indicate mean (horizontal bar) and standard deviation. B, Individual plots showing the change in CD8⁺ T-cell tetramer binding following immunizations for peripheral blood mononuclear cells of (i) CMV-seropositive unique patient number (UPN) 212 vaccinated with 0.5 mg Tet-CMV + PF03512676 adjuvant; (ii) CMV-seropositive UPN 195 vaccinated with 2.5 mg PADRE-CMV + PF03512676 adjuvant; (iii) CMV-seropositive UPN 159 vaccinated with 0.5 mg CMV PADRE + PF03512676 adjuvant. Solid circles show pp65_495-503–specific tetramer binding; open circles indicate binding to HIVgag_77-85 (control) tetramer. Arrows indicate the day of vaccination.

Figure 3.

Kinetics of vaccination responses. Longitudinal profiles of ex vivo pp65_495-503–specific tetramer binding (expressed in percentages) for trial A (left plots) and trial B (right plots). A, Cytomegalovirus (CMV)^<0.2% subjects. P value indicates the difference between trial A and trial B, calculated by Wilcoxon rank-sum test. B, CMV-seropositive subjects who had at baseline ≥0.2% pp65_495-503 tetramer binding.

Figure 4.

Vaccine memory responses. For each subject, in vitro stimulation (IVS) was performed at 3 or more time points and always included baseline blood draws. A, Individual fluorescence-activated cell sorting plots showing the levels of pp65_495-503 CD8⁺ T cells after IVS, using peripheral blood mononuclear cells (PBMCs) of volunteers who did not have an ex vivo response to the cytomegalovirus (CMV) peptide vaccines. The upper plots refer to IVS performed with PBMC of representative CMV-seropositive unique patient number (UPN) 196 vaccinated with 2.5 mg PADRE-CMV + PF03512676 adjuvant. The tetramer used and the number of injections performed are reported on the y-axes and top plot, respectively. The lower plots refer to IVS performed with PBMC of CMV-seronegative UPN 172 vaccinated with 0.5 mg PADRE-CMV + PF03512676 adjuvant and report the levels of interferon γ (IFN-γ) produced by CD8⁺ T cells using pp65_495-503 peptide as stimulant during intracellular cytokine staining. The number of injections is reported on the top plot. B, The graph shows the median levels of CD8⁺ T cells specific for either the pp65_495-503 (solid symbols) or the HIVgag_77-85 (control, open symbols) tetramers in function of vaccine injections in 4 CMV-seronegatives and 4 CMV^+<0.2% subjects from trial B, who failed to show an ex vivo CMV peptide vaccine response (Table 3). P value was calculated by Wilcoxon rank-sum test to assess the significance of postvaccination binding difference between pp65_495-503 and control (HIVgag_77-85) tetramer in the IVS-expanded CD8⁺ T cells.