in clinical trials to date have been recombinant poly-protein vaccines, formulated with a 76 variety of lipid-based adjuvants primarily aimed at eliciting CD4 + T cell responses (reviewed 77 in [19] [20] [21] ), but these studies have fallen short of demonstrating efficacy in either a prophylactic 78 or therapeutic setting. Leishmania as an intracellular pathogen may also be targeted for 79 immune destruction by effector mechanisms of CD8 + T cells (including IFNg production and 80 granzyme / granulysin release 22 ) and CD8 + T cell responses have been associated with 81 vaccine-induced protection in animal models [23] [24] [25] [26] . Vaccines designed to generate CD8 + T 82 cell responses require the ability of antigen delivery into the endogenous processing pathway. 83 This is achieved either by facilitating cross-presentation (e.g. using liposomal delivery) or 84 through endogenous protein synthesis (e.g. naked DNA or viral vectors; so called "third 85 generation" vaccines). 86
We recently described a novel third generation adenovirus-vectored vaccine (ChAd63-KH). 88
ChAd63-KH is based on a well-characterised simian adenovirus backbone (ChAd63), 89 extensively tested in human volunteers and shown to have an excellent safety record 27 . 90
ChAd-vectored vaccines induce potent CD8 + and CD4 + T cell responses and antibodies in 91 humans and are amenable to scalable manufacture at GMP. ChAd63-KH encodes two 92
Leishmania antigens, kinetoplastid membrane protein-11 and hydrophilic acylated surface 93 protein B (K; KMP-11 and H; HASPB), both with prophylactic and therapeutic vaccine 94 efficacy when used as monovalent vaccines in pre-clinical animal models (mouse, hamster or 95 dog) 24,25,28 . KMP-11 is a highly conserved membrane protein expressed in promastigotes and 96 amastigotes of all Leishmania examined to date and is rich in CD8 + T cell epitopes 23 . 97 HASPB is expressed by infective metacyclics and amastigotes 29 and has conserved N and C 98 termini flanking polymorphic repeats. These repeats differ in copy number and arrangement 99 across isolates of L. donovani 30 although the functional significance of this is unknown. To 100 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 duration. Participants were patients diagnosed with persistent PKDL aged between 18-50 126 (adults) or 12-16 (adolescents) . The diagnosis of a case of persistent PKDL was based on a 127 typical distribution of the skin rash for a duration of six months or more, a temporal 128 relationship treated kala-azar, a reactive serology test and exclusion of other skin condition. 129 PKDL lesions were defined per protocol as grade 1 (scattered maculopapular or nodular 130 lesions, mainly around the mouth), grade 2 (dense maculopapular or nodular rash covering 131 most of the face and extending to chest, back, upper arms and legs or grade 3 (dense 132 maculopapular or nodular rash covering most of the body, including hands and feet.) 33 . 133
Inclusion criteria included: uncomplicated PKDL of > 6 month's duration; availability for the 134 duration of the study; otherwise good health as determined by medical history, physical 135 examination, results of screening tests and the clinical judgment of a medically qualified 136 Clinical Investigator; negative for malaria on blood smear; judged able and likely to comply 137 with all study requirements; willing to undergo screening for HIV, Hepatitis B and Hepatitis 138 C; for females only, willing to undergo urinary pregnancy tests on the day of screening, on 139 the day of vaccination (prior to vaccination) and 7 and 42 days after vaccination. Exclusion 140 criteria included: mucosal or conjunctival PKDL; treatment for PKDL within 21 days; 141 negative for antibodies in the RK39 strip test; receipt of a live attenuated vaccine within 60 142 days or other vaccine within 14 days of screening; administration of immunoglobulins and/or 143 any blood products within the three months preceding the planned administration of the 144 vaccine candidate; history of allergic disease or reactions likely to be exacerbated by any 145 component of the vaccine or a history of severe or multiple allergies to drugs or 146 pharmaceutical agents; any history of severe local or general reaction to vaccination; for 147 females only, pregnancy, less than 12 weeks postpartum, lactating or willingness/intention to 148 become pregnant during the study and for 3 months following vaccination; seropositive for 149 hepatitis B surface antigen (HBsAg) or Hepatitis C (antibodies to HCV); any clinically 150 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 (depending on cohort). An independent data safety and monitoring board (DSMB) meeting 176 was held at the end of each cohort to review data and provide advice to the Sponsor regarding 177 continuation of the trial. Clinical and biochemical test abnormalities were graded according 178 to the protocol, based on NIH guidelines. Treatment for adverse events was provided as 179 required. The natural history of PKDL in Sudan indicates that patients with disease persistent 180 for greater than six months are unlikely to self cure 34 . A final end point for clinical response 181 was scheduled to be made between day 42 and day 90 (see Results) Patients with less than 182 75% improvement were offered standard treatment with AmBisome® (2.5mg/kg/day for 20 183 days), those with between 75-90% improvement were offered conservative treatment or 184
AmBisome®, and those with greater than 90% clinical improvement were deemed to not 185 require further treatment. Standard treatment with AmBisome® (20 days; 2.5mg/kg/day) 186 was provided in hospital and patients were confirmed as clinically cured at the end of 187 treatment. Some patients defaulted from scheduled visits and were evaluated and treated at 188 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. . https://doi.org/10. 1101 /2020 sequencing libraries with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit. 201 Libraries were barcoded, purified with 2.5 X Agencourt AMPure XP Magnetic Beads 202 (Beckman Coulter) and then quantified using Ion Library TaqMan Quantitation Kit on a 203
QuantStudio 5. Libraries were diluted to a concentration of about 50 pMol and pooled in 204 groups of 8 for sequencing on Ion PI Chips. Chips were loaded using the Ion Chef System 205 and the IonPI Hi-Q Chef Kit. Sequencing was performed on an Ion Proton Sequencer using 206
Ion PI Hi-Q Sequencing 200 Kit. Data will be deposited in GEO at publication. 207 208 Differential gene expression analysis was performed using DeSeq2 35 . After count data 209 normalization, differential gene expression analysis was performed using pooled day 0 data 210 from the three study cohorts as the baseline for all contrasts. Enrichment of blood 211 transcription modules at each time point in the different groups was assessed with the tmod R 212 package 36 , using as an input the lists of differentially expressed genes ranked by the p-value 213 after multiple test correction, as computed by DeSeq2. Significance of module enrichment 214 was assessed using the CERNO statistical test (a modification of Fisher's combined 215 probability test) and corrected for multiple testing using the Benjamini-Hochberg correction. 216
In order to identify the modules with highest correlation to the clinical response, the Z-score 217 of each module was used to train a 1-dimensional Logistic Regression model. After 100 218 bootstraps the modules were ranked according to the average prediction score. Data analyses 219 were performed by python scripts using the scikit-learn python library 37 . Gene set 220 enrichments were performed in EnrichR 38 and pathway analysis was conducted using IPA 221 (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
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Ex vivo re-stimulation of frozen PBMC to elicit vaccine-induced CD8 + T cell responses was 227 performed using Multiscreen IP ELISpot plates (Millipore), human IFNg SA-APL antibody 228 kits (Mabtech) and BCIP-NBT-plus chromogenic substrate (Moss Inc) as previously 229 described 32 . Peptide re-stimulation was restricted to a peptide pools corresponding to the 230 entire KMP-11 sequence and the N terminal conserved domain of HASPB1 (pools 1 and 2 32 ) 231 due to limitations in cells obtained from patients. Responses to medium only negative 232 controls were subtracted and responses >50 SFC / million above the pre-vaccination response 233
were regarded as positive. Antibody responses will be reported at a later date due to 234 inaccessibility of trial samples at the current time due to COVID-19. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
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Thirty-nine patients were screened for eligibility between November 2016 and April 2019 253 ( Figure 1 ) and 24 patients with the demographic and baseline biochemical and hematological 254 characteristics shown in Table 1 and Table S1 were enrolled in the study. In each of the 255 adult cohorts, there were six patients with grade 1 PKDL and two patients with grade 2 256 PKDL. In the adolescent cohort, there were two patients with grade 1 PKDL, five patients 257 with grade 2 PKDL and one patient with grade 3 PKDL. Median duration of PKDL in the 258 study population was 30 months (range 6 -180 months). In the adult high dose, adult low 259 dose and adolescent cohorts respectively, 4 of 8 (50%), 6 of 8 (75%) and 3 of 8 (37.5%) 260 patients had had PKDL for >12 months duration. Twenty two of all 24 patients were 261 followed up at the scheduled D90 visit, and 5 of 8 patients in the adolescent cohort were 262 followed up to their D120 scheduled visit (Figure 1) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. . https://doi.org/10.1101/2020.08.26.20180901 doi: medRxiv preprint SUSARs reported (Table S1) to study completion resolved their PKDL lesions without the need for chemotherapy. 294
295
Immune responses in the patient cohort were assessed using whole blood transcriptomic 297 analysis (WBTA), comparing pre-vaccination blood to blood taken at 1, 3 and 7 days post 298 vaccination. Differentially expressed genes were identified (Table S2 ) and used to identify 299 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020. . https://doi.org/10.1101/2020.08.26.20180901 doi: medRxiv preprint transcriptional modules significantly associated with vaccination (Figure 4 and Table S3 ). 300
These data identified three key features of the response. First, modules associated with an 301
anti-viral signature and with dendritic cell activation showed a marked dose dependence, 302 being minimal in patients receiving low dose vaccine. Second, there was a near equivalence 303 of these innate responses in adults and adolescents receiving high dose vaccine. Third, 304
modules associated with B cell responses were prominent only in adolescents. As 305 approximately 30% of patients resolved their PKDL over the follow up period, we sought to 306 identify potential predictors of resolution. We used a logistic regression model to identify 307 potential predictors of patients with >90% clinical resolution, using the Z scores associated 308
with the blood transcription modules (Figure 4) . This analysis revealed 11 modules ( 309 Predictive modules sheet in Table S3 ), focused on monocyte and dendritic cell attributes, 310 identified to have highest predictive value for clinical response. Among these 11 modules, 311 two were also identified as differentially expressed: LI.M139 (lysosomal/endosomal proteins) 312 and LI.M118.0 (enriched in monocytes) ( Table S3) . 313
We also analysed differentially expressed genes identified at day 1 post vaccination with 315 7.5x10 10 vp by Ingenuity Pathway Analysis and for gene set enrichment (using EnrichR). 316
Comparative pathway analysis of differentially expressed genes in adults (374 UP; 108 317 DOWN) and adolescents (510 UP; 439 DOWN) showed a high degree of concordance in 318 predicted upstream regulators, e.g. IFNG (z score of 9.169 vs 8.847 for adults vs. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. . https://doi.org/10.1101/2020.08.26.20180901 doi: medRxiv preprint (Table S4) , in keeping with the results of the modular analysis described above. Thus, 324 patients with PKDL appear to mount effective innate cellular responses to ChAd63-KH. 325 326 CD8 + T cell response following vaccination 327
IFNg production by CD8 + T cells was measured using ELISpot following re-stimulation with 328 peptide pools. The frequency of CD8 + T cells producing IFNg specifically in response to 329 KMP-11 (pool 1) was not significantly different between high and low dose vaccinated adults 330 nor between adult and adolescent cohorts ( Figure 5A) . Overall ELISpot responder frequency 331 to this peptide pool was 75% (18/24), with peak responses in responders ranging from 86-332 3766 SFC/million PBMC (mean 509; 95% CI 93-926) ( Figure 5B ). Responses were 333 comparable in frequency and magnitude to those seen previously in healthy UK volunteers 334 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
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The rationale for using vaccines for therapeutic benefit or for post exposure prophylaxis is 349 not new and has been evaluated for a variety of chronic viral infections including HIV 43 and 350 HPV 44 as well as in cancer 45 . Whilst some chronic viral infections may subvert immunity to 351 such an extent to pose a barrier to therapeutic vaccination 46 , recent evidence suggest that such 352 limitations to vaccine efficacy can be overcome 47 . Evidence also suggests leishmaniasis 353 patients may respond well to therapeutic vaccination. For example, protective immunity is 354 readily reactivated after drug cure 11 ; virally-vectored antigen delivery generates effector 355 CD8 + T cells and therapeutic benefit in rodents 31,48 ; and there have been encouraging data 356 from human immunochemotherapy trials in leishmaniasis patients 16, 17 . 357 358 PKDL in Sudan has a complex natural history 5-7 . In most cases it emerges within 3-6 months 359 of the cessation of treatment for VL, reminiscent of an immune reactivation disease 5 although 360 cases also occur during and even in the absence of prior VL 4,49 . In a study of the natural 361 history of PKDL in 134 children younger than 14 34 , 84% of patients showed spontaneous 362 remission of their disease with a mean (SD) of 9.7 (4.7) months. For the remaining 16% (21 363 patients), duration of PKDL was 16.6 (5.5) months, with over half of these cases showing 364 either no change or worsening of PKDL during the first 12 months. Grade or severity of 365 PKDL did not appear to influence the duration of PKDL and unless disease was very severe, 366 these patients did not require treatment 34 . Although formal time to event data are not 367 available, and other age groups have not been studied systematically, current clinical practice 368 in Sudan is based on the premise that patients with persistent PKDL for six months or longer 369 duration are not expected to rapidly self-resolve their lesions, and such patients are therefore 370 provided with the standard of care, a protracted course of liposomal amphotericin B 371 (AmBisomeâ; 2.5mg/kg/day for 20 days). Hence, as for other studies evaluating new drug 372 regimens for PKDL 16,50,51 , patients with PKDL of greater than six months duration were 373 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 to be determined and the small sample size and variable demographics suggest this finding 399 needs confirmation with a larger sample size and through more detailed phenotypic and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. The funders played no role in study design or decision to publish. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
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The copyright holder for this this version posted September 1, 2020. . https://doi.org/10.1101/2020.08.26.20180901 doi: medRxiv preprint The CONSORT diagram reports attendance at scheduled in-patient and out-patient visits. 457
Clinical data was also collected for some individuals at additional unscheduled visits as 458 shown in Figure 3 . Kit. Each column represents a different time-point (days 1, 3, and 7) after vaccination in the 476 three study groups (low-dose adults, high-dose adults, high-dose adolescents). Significantly 477 enriched immune-related modules were identified applying the CERNO test on the adjusted 478 p-value-ranked lists of genes generated by DeSeq2 (see Table S2 for module gene lists). 479 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020 . . https://doi.org/10.1101 /2020 Modules are represented by bars in which the proportion of significantly upregulated and 480 downregulated genes is shown in red and blue, respectively. The gray portion of the bar 481 represents genes that are not significantly differentially regulated. The significance of module 482 activation is proportional to the intensity of the bar, while the effect size is proportional to its 483 width. Table S1 . Demographic, baseline and AEs summary for study participants. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. The CONSORT diagram reports attendance at scheduled in-patient and out-patient visits.
Clinical data was also collected for some individuals at additional unscheduled visits as 683 shown in Figure 3 . 684 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. . https://doi.org/10.1101/2020.08.26.20180901 doi: medRxiv preprint 755 . CC-BY 4.0 International license It is made available under a perpetuity.
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The copyright holder for this this version posted September 1, 2020. Figure 5 . CD8 + T cell response to vaccination with ChAd63-KH 813 PBMC from patients collected from d7-d90 post vaccination were stimulated with peptide 814 pools representing the entire KMP-11 sequence (P1) and the HASPB N terminus (P2). The 815
number of IFNg producing cells / million PBMC was determined by ELISpot. A. Peak 816 response by cohort to P1. B. Pre-vaccination and peak post vaccination response per patient 817 to P1 for low dose adult (green), high dose adult (blue) and high dose adolescents (orange); C 818 and D. Comparison between patients in this trial (LEISH2a) and healthy UK volunteer 819 responses (LEISH1) for response to P1 (C) and P2 (D) . Data for LEISH1 is taken from 32 . 820
Box and whisker plots indicate median, 25 th -75 th quartiles, mix/max values and individual 821 patient data points. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint
The copyright holder for this this version posted September 1, 2020. . https://doi.org/10. 1101 /2020 