A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH

Abstract

Background: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells.

Methods: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry.

Findings: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects.

Conclusion: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL.

Trial registration: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359).

Fig 1. CONSORT diagram for LEISH1 first-in-human clinical trial.

Fig 2. Summary of local and systemic adverse events in LEISH1.

A. Percentage of subjects vaccinated with low or high dose ChAd63-KH with at least one grade 1 (open bar) or grade 2 (grey bar) adverse events. B. Number of local and systemic adverse effects in subjects vaccinated i.m. with 1x10¹⁰ vp (open bar) or 7.5x10¹⁰ vp (grey bar). Data are shown as median ± interquartile range. C. Individual peripheral blood lymphocyte counts in low dose (open circles) and high dose (black circles) subjects pre- vaccination and at 24h and 14 days post-vaccination. Significant lymphopenia was defined as >25% reduction in lymphocyte count.

Fig 3. Innate immune response to ChAd63-KH vaccination.

Whole blood from high dose subjects was collected before vaccination and at 24h post vaccination and processed for RNA-Seq. A. Volcano plot showing Log2FC in gene expression (y axis) against signal intensity (Log2CPM). B. Principle component analysis showing clustering of pre- (black, by subject number) and post- (red, by subject number) vaccination samples. C. Frequency of naïve and resting memory CD4⁺ T cells, monocytes and activated DCs pre and post vaccination, as determined by CIBERSORT analysis. D. Module level analysis comparing gene representation pre and post vaccination. Colour code represents proportion of genes significantly changed (over-represented, red; under-represented, blue) for each of the 28 modules described by Chaussabel et al [47].

Fig 4. Interferon signatures associated with ChAd63-KH vaccination.

A. Scatter plot showing magnitude of differential expression for the 103 genes commonly regulated in low and high dose subjects. B and C. DE genes associated with the IPA canonical IFN pathway in low dose (B) and high dose (C) subjects. Degree of fold change is shown in red scaling. D. Radar plot showing fold change in gene expression for genes identified by Quinn et al as related to antigen presentation (module C2; [44]). Data are shown for low dose (red) and high dose (blue) subjects, as well as for mice immunized with ChAd3 (yellow) and ChAd63 (green). Mouse data originated from Quinn et al [44].

Fig 5. CD8⁺ T cell responses to ChAd63-KH vaccination.

A. Schematic to illustrate coverage of the KH antigen by the CD8 selective peptide pools (Pools 1, 2, 3.1, 3.2, 3.3 and 4) and CD4/8 15mer peptide pools (Pools A, B and C) used in this study. B. ELISPOT response to ChAd63-KH vaccination over time by individual low dose (Don 1, 3, 4, 6, 10; open symbols) and high dose (closed symbols) subjects. Data represent sum of response to all peptide pools at each time indicated. Average responses for low and high dose subjects are shown in S3 Fig. C. Peak response to each peptide pool for low (open circles) and high (black circles) dose subjects. Median responses for high (solid line) and low (dotted line) dose subjects are also shown. D. Breadth of response, reflecting number of peptide pools recognized by each subject group. E. Correlation between breadth of response and sum of total response (R² = 0.3437, p = 0.0134).

Fig 6. Intracellular cytokine production by CD8⁺ T cells.

A. IFNγ, TNF and IL-2 responses at day 28 post vaccination to individual peptide pools. Box and whisker plots showing frequency of cytokine producing cells as % of total CD8⁺ T cells. Data are pooled for all low and high dose subjects (n = 20). B. Cytokine producing CD8⁺ T cells producing one, two or three cytokines are shown by individual donor for each peptide pool. Low dose (open circles) and high dose (closed circles) subjects are shown separately. C. Heat map to show frequency of low dose (left; n = 5) and high dose (right; n = 15) subjects responding (at a cut-off of 0.05%) with single, dual or triple cytokine production after stimulation with each peptide pool.

Fig 7. Antibody and CD4⁺/CD8⁺ T cell responses to ChAd63-KH vaccination.

A. ELISPOT response to ChAd63-KH vaccination over time for individual low dose (Don 1, 3,4,6,10, open symbols) and high dose (closed symbols) subjects. Data represent sum of response to peptide pools A, B and C at each time indicated. B. Peak response to each peptide pool for low (open circles) and high (black circles) dose subjects. Median responses for high (solid line) and low (dotted line) dose subjects are also shown. C. Antibodies specific for the rKH protein were assayed by ELISA. Data are shown for all subjects at the indicated times post vaccination.