MVA vaccine encoding CMV antigens safely induces durable expansion of CMV-specific T cells in healthy adults

Abstract

Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933.

© 2017 by The American Society of Hematology.

Figures

Figure 1.

Triplex vaccine construct and vaccination regimen. (A) Vaccine characteristic: schematic representation of Triplex vaccine. Direct repeats have been previously described. FL1 and FL2 are flanking (FL) DNA of deletions II and III. Plasmid mH5-pp65-pLW51 and mH5-IEfusion-pZWIIA structures and the modified H5 promoter have been detailed elsewhere., TK, thymidine kinase gene of MVA. Arrows show direction of transcription. (B) Vaccination regimen: 2 injections of Triplex vaccine were administered at each DL as indicated. Postvaccination follow-up days are shown on the bar. Letters pointed by the blue arrows detail laboratory and immune evaluations, clinical and AE assessment. In detail, a, urine pregnancy test (female subjects); b, metabolic and hematologic panels; c, physical examination; d, electrocardiogram and troponin test; e, AE monitoring; f, MVA vector persistence measurements; g, immunological assays; h, CMV serology; I, HIV, hepatitis B and C tests; l, HLA typing at A and B loci. For b, f, g, h, i, and l, drawing blood was required.

Figure 2.

pp65-specific T-cell expansion. (A) Postvaccination levels of pp65 CD8 and CD4 T cells. In the box plots, red circles indicate DL1; green circles indicate DL2; blue circles indicate DL3. Boxes cover central 50% of observations, and the central bars show median; whiskers extend to at most 1.5 times box length with more extreme observations shown individually (colored circles). Colored lines represent means for each DL, and black dotted lines show the fitted means (referred as “Model” in the upper right box) on a square-root scale from a piecewise linear GEE model, with a change in slope at day 42 and no DL distinction. Thinner lines indicate the responses profiles for the 3 CMV seronegatives enrolled in the study. UPN 14 (DL2) and 18 (DL3) remained CMV seronegative until day 180, but tested CMV seropositive at day 360. UPN 13 (DL2) received smallpox vaccination. Arrows indicate Triplex vaccine injection. (B) Representative dot plots and gating hierarchy. In the top row, the primary gate set on lymphocytes by use of forward and side scatter is shown. Histogram plots indicate (from left to right) the CD8 (fluorescein isothiocyanate [FITC] conjugated) gating, and the subsequent gating of CD8+ CD137+ (allophycocianin [APC] conjugated) T cells from CMV-seronegative UPN 14, stimulated with the pp65 library on day 180 post-Triplex vaccination (as indicated by the arrow). In the upper right gate of each dot plot, the percentage of CD8+ CD137+ T cells in pp65 stimulated (upper dot plots) and unstimulated (lower dot plots) PBMC samples are shown collected at the post-Triplex vaccination days indicated.

Figure 3.

CMV-specific T-cell responses. (A) IE1-exon4- and IE2-exon5-specific T-cell responses. UPN 24 (top plot) and UPN 9 (lower plot) represent longitudinal profiles, showing expansion of the 3 CMV antigens expressed by Triplex. Two injections of Triplex were administered on days 0 and 28. (B) CD8+ pp65495-503-specific T-cell levels. Box plots cover central 50% of observations, and the central bars show median. Whiskers extend to at most 1.5 times box length, and individual observations are shown with colored circles. Red circles indicate subjects from DL1; green indicates subjects from DL2, and blue indicates subjects from DL3. The black line shows the fitted means on a square-root scale from a piecewise linear GEE model, with a change in slope at day 42 and no DL distinction.

Figure 4.

CMV-specific memory phenotypes and MVA vector-specific responses. (A) Memory phenotype of CMV-specific T cells following Triplex vaccination. The top panel shows representative FACS plots (from UPN 13; gray boxes in the subsequent panels indicate the selected time point) relative to the gating hierarchy used for the memory phenotype analysis of IFN-γ producing T cells. From left to right, lymphocytes gated by use of forward and side scatter; histogram plots indicating the CD3 (peridinin chlorophyll protein Cychrome 5 [PerCP Cy5.5] conjugated) and the CD8 (violet laser excited coumarin dye [V450] conjugated) gating; subsequent gating of CD8+ IFN-γ+ (APC conjugated) T cells unstimulated or stimulated with the pp65 library (as specified on the plot title), and respective percentages shown in the upper right gate of each contour plot. By gating pp65-specific CD8+ IFN-γ+ T cells, 4 subpopulations were identified according to the expression of CD28 and CD45RA (far right dot plot). CD45RA+ CD28+ cells were classified as naive (upper right quadrant); CD45RA− CD28+ cells were classified as central memory (TCM, lower right quadrant), and CD28− cells were classified as effector. Within the effector T-cell group, 2 subpopulations were identified: CD45RA− CD28− (TEM, T effector memory, lower left quadrant) and CD45RA+CD28− (TEMRA, revertant T-effector memory re-expressing CD45RA, upper left quadrant) T cells. The middle panel of line graphs shows the longitudinal profiles of levels of CMV-specific CD8+ and CD4+ T cells producing IFN-γ in CMV-seronegative UPN 13, UPN 14, and UPN 18 following stimulation with the peptide library indicated in the legend. The lower panel of line graphs shows the percentages of the respective memory phenotypes indicated in the legend for CMV-specific CD8+ IFN-γ+ T cells. Memory phenotypes were analyzed when CMV-specific IFN-γ production by T cells in response to a CMV-specific library was ≥0.2%. UPN 13 had been vaccinated against smallpox. Arrows show time of vaccinations (days 0 and 28). The star symbol indicates that UPN 14 and UPN 18 seroconverted and were tested to be CMV seropositive on day 360. (B) T-cell response to the MVA vector. Box plots show levels of vaccinia-specific CD8 T cells on a square-root scale. Colored lines indicate DL and the fitted means are shown as detailed in Figure 2. (C) MVA-specific neutralizing antibodies. The right plot shows MVA specific NT for DL2 and DL3 cohorts, expressed as inhibition dilution (ID50), which is the serum dilution that caused 50% reduction in fluorescence. Box plots cover central 50% of observations, and the central bars show median; whiskers extend to at most 1.5 times box length. All patients are individually shown by a line, as specified in the legend; “pre72” indicates that the subject received smallpox vaccination being born before 1972, during the compulsory smallpox vaccination campaign. The percentage of infection neutralization (ID inhibition dilution) was calculated as follows: (1 − [percentage of fluorescence in Epstein-Barr virus transformed lymphoblastoid cell line cells incubated with serum from vaccinated subject/[percentage of fluorescence in untreated Epstein-Barr virus transformed lymphoblastoid cell line controls]) × 100. Dilutions for each sample ranged from 1:20 to 1:1000. The ID50 is the serum dilution that caused 50% reduction in fluorescence and was calculated by determining the linear slope of the graph plotting ID versus serum dilution by using the next higher and lower ID values that were closest to 50% neutralization.