A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults

Vaccine xxx (xxxx) xxx

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A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults Malcolm S. Duthie a,b,c,⇑, Aude Frevol a, Tracey Day a, Rhea N. Coler a,c,d, Julie Vergara a, Tom Rolf a, Zachary K. Sagawa a, Anna Marie Beckmann a,c, Corey Casper a, Steven G. Reed a,b,c a

Infectious Disease Research Institute, Seattle, WA, USA Host Directed Therapeutics, Seattle, WA, USA c Department of Global Health, University of Washington, Seattle, WA, USA d PAI Life Sciences Inc., Seattle, WA, USA b

a r t i c l e

i n f o

Article history: Received 22 October 2019 Received in revised form 9 December 2019 Accepted 20 December 2019 Available online xxxx Keywords: Leprosy Vaccine Adjuvant Immunologic Safety

a b s t r a c t Healthy United States-based adult volunteers with no history of travel to leprosy-endemic countries were enrolled for the first-in-human evaluation of LepVax (LEP-F1 + GLA-SE). In total 24 volunteers participated in an open-label clinical trial, with 21 receiving three injections of LepVax consisting of either 2 mg or 10 mg recombinant polyprotein LEP-F1 mixed with 5 mg of the GLA-SE adjuvant formulation. LepVax doses were provided by intramuscular injection on Days 0, 28, and 56, and safety was evaluated for one year following the final injection. LepVax was safe and well tolerated at both antigen doses. Immunological analyses indicated that similar LEP-F1-specific antibody and Th1 cytokine secretion (IFN-c, IL-2, TNF) were induced by each of the antigen doses evaluated within LepVax. This clinical trial of the first defined vaccine candidate for leprosy demonstrates that LepVax is safe and immunogenic in healthy subjects and supports its advancement to testing in leprosy-endemic regions. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Leprosy (Hansen’s disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae. The estimated global new case incidence rate is approximately 220,000 per year. Nerve involvement in leprosy patients can manifest in the form of sensory and/or motor neuron damage that can advance to cause disability and disfigurement. This nerve damage likely involves a complicated interplay of both host immunity and bacterialmediated events [1,2]. Although bacterial cure can be achieved with the multidrug therapy (MDT) cocktail that has now been provided free of charge

Abbreviations: AE, adverse event; BCG, M. bovis bacillus calmette-guérin; ELISA, enzyme linked immunosorbent assay; GLA, glucopyranosyl lipid adjuvant; IFNc, interferon gamma; IL, interleukin; MB, multibacillary; MDT, multidrug therapy; MSD, mesoscale discoveries; PB, paucibacillary; PBS, phosphate buffered saline; SAE, serious adverse event; SE, stable emulsion; Th1, T helper 1-like cells; TLR, tolllike receptor; WBA, whole blood assay; WHO, world health organization. ⇑ Corresponding author at: Host Directed Therapeutics (HDT), 1616 Eastlake Ave E., Suite 280, Seattle, WA 98102, USA. E-mail address: [email protected] (M.S. Duthie).

for registered leprosy patients for many years, leprosy remains as a public health problem in many regions. Declines in global incidence and drive toward ‘elimination’ as a global health problem by the year 2000 have now levelled off and it is widely believed that a large number of cases go unreported [3,4]. Indeed, epidemiologic data indicate that transmission continues and the disease is slowly re-emerging in many countries that previously reported elimination and active case finding studies typically find up to six times more cases than would be anticipated based upon reported numbers [5–7]. Thus, new control strategies are merited. The current strategies for preventing M. leprae transmission and disease rely on the provision of MDT to infected patients or postexposure chemoprophylaxis of at-risk persons. These strategies are, however, limited in their durability by the drug half-life and efficacy in already infected people. Unlike drug treatment, vaccines could be used to provide active and sustained protection in both uninfected and infected individuals. An effective vaccine could be a critical element of control. Indeed, although no specific vaccine against leprosy is currently available, immunization with the Mycobacterium bovis BCG vaccine, which was developed for tuberculosis, provides some variable degree of protection. Protection

https://doi.org/10.1016/j.vaccine.2019.12.050 0264-410X/Ó 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

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afforded by BCG vaccination is highest in younger individuals and wanes over time [8–10]. While a BCG re-immunization strategy has not been effective against TB and WHO guidelines do not support it for leprosy [11–13], because some studies indicate that multiple BCG vaccinations can enhance protection some countries have implemented BCG re-immunization of leprosy patients or contacts [14,15]. Although immunoprophylaxis by BCG vaccination of contacts of newly diagnosed leprosy patients conferred 56% protection in a cohort study in Brazil [16], meta-analyses of numerous clinical trials estimated the protective effect of BCG in preventing leprosy to be modest (26% and 41%, respectively) and the persistence of leprosy in regions with good BCG coverage indicates that additional, or alternative, vaccines are required [17,18]. Through recombinant expression and preclinical development, we prioritized M. leprae antigens recognized by T cells of leprosy patients and their contacts for leprosy vaccine development, resulting in the antigen LEP-F1. LEP-F1 (known as ML89 during its preclinical development) was formed by the tandem linkage of four M. leprae open reading frames encoding the proteins ML2531, ML2380, ML2055, and ML2028 [19]. When formulated with a synthetic Toll-like Receptor (TLR) 4 adjuvant (glucopyranosyl lipid adjuvant; GLA) in a stable oil-in-water emulsion (GLA-SE) to generate the vaccine candidate we call LepVax, potent LEP-F1-specific Th1 responses can be induced in immunized mice [19,20]. Furthermore, provision of LepVax to already M. lepraeinfected armadillos delays the onset of nerve conduction deficits and reduces their severity [19]. In this first-in-human trial we evaluated the safety and immunogenicity of LepVax (LEP-F1 + GLA-SE) in healthy uninfected subjects. To do so, we enrolled subjects in a leprosy nonendemic country (the United States) with a primary objective of determining the safety and tolerability of two distinct doses of the vaccine candidate. The secondary objective was to assess the immunogenicity of the vaccine candidate by evaluating the durability of Th1 cell-mediated responses to the LEP-F1 protein. 2. Materials and methods 2.1. Ethical conduct of the study We performed a single center Phase 1, open-label clinical trial designed to evaluate the safety, tolerability, and immunogenicity of LepVax (LEP-F1 + GLA-SE) following intramuscular administration in healthy adult subjects. This study was registered in ClinicalTrials.gov (Identifier NCT03302897) and conducted in accordance with the E6 Guidance of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) ‘‘Good Clinical Practice: Consolidated Guidance” and the U.S. Code of Federal Regulations governing the protection of human subjects (21 CFR 50), Institutional Review Boards (21 CFR 56), and the obligations of clinical investigators (21 CFR 312). The study protocol and informed consent form (ICF) were approved by Midlands Independent Review Board (Overland Park, KS). 2.2. Participants and treatments The subjects were recruited from Madison, Wisconsin, and the surrounding area and were healthy male and female adults
18 years and 55 years of age with no history of travel to areas endemic for leprosy. Female subjects of childbearing potential were required to have a negative serum pregnancy test at screening and a negative urine pregnancy test on the day of each study injection. All potential subjects were recruited after obtaining a signed ICF prior to performing screening procedures. At screening,

normal (or not clinically significant as determined by the investigator and the medical monitor) hematologic and serum chemistry values and negative serological tests for human immunodeficiency virus (HIV) 1/2, hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV) were required for inclusion. A total of twenty four subjects were then sequentially enrolled into one of two treatment groups (Fig. 1A). The first treatment group received a 2 mg LEP-F1 dose per injection that, when no stopping rules were observed by day 35, increased to 10 mg LEP-F1 per injection for the second treatment group. The GLA-SE adjuvant formulation dose was maintained at 5 mg per dose. LEP-F1 (P/N 0237 Lot 063I0816) and GLA-SE (P/N 0238 Lot 16 K002; labeled as AP 10-201) were used (both from IDRI). Subjects in both treatment groups received a total of three intramuscular study injections in the deltoid, one injection on each of days 0, 28, and 56. The first and third study injections were placed in the left arm and the second study injection was in the right arm. 2.3. Safety parameters General safety was evaluated at baseline (i.e., determining suitability for trial entry and providing informed consent) and on Days 0 (day of first study injection), 28, 56, 84, 168, and 421 for each subject. The primary safety endpoints assessed were the proportion of subjects with adverse events (AEs), serious adverse events (SAEs), local injection-site reactions, and specific systemic reactions to the study injections from Day 0 to Day 84. The occurrence of SAE and adverse events of special interest (AESI) continued to be monitored through Day 421. Adverse events were graded by severity (Grade 1 to 4) and by relationship to study injection (unrelated, unlikely, possibly, probably, or definitely related) according to predetermined definitions. Local injection site reactions were assessed on the day of study injection administration (prior to injection and 60 min after injection) and on the second and seventh days after study injection administration by grading pain, erythema, and induration as Grade 1 (mild), Grade 2 (moderate), Grade 3 (severe), or Grade 4 (potentially life-threatening). Solicited systemic reactions were defined as the occurrence of headache, arthralgia, chills, loss of appetite, fever, fatigue, and myalgia in the seven days following each study injection, with symptoms similarly graded as Grade 1 (mild), Grade 2 (moderate), Grade 3 (severe), or Grade 4 (potentially lifethreatening). Data were collected by each subject by completing memory aid booklets that were distributed to collect local site of injection, solicited systemic adverse event information, and oral temperature over the 6-day period following each injection. Vital signs and safety laboratory evaluations encompassing hematology (hemoglobin, WBC count, platelet count) and serum chemistry parameters (sodium, potassium, creatinine, BUN, ALT, alkaline phosphatase, total bilirubin) were measured in blood samples collected at screening, and seven days after each injection (days 7, 35, and 63). 2.4. Evaluation of anti-LEP-F1 immune responses Blood was collected from each subject by venipuncture at baseline (day 0), day 7, 35 and 63. To determine anti-LEP-F1 specific antibody responses, serum was prepared and stored at 70 °C until the IgG antibody isotype was measured by ELISA. Briefly, High Binding 384-well ELISA plates (Corning, Corning, NY) were coated with 1 lg/ml LEP-F1 antigen in ELISA Coating buffer (Invitrogen, CA) and blocked with 1% BSA-PBS. Then, in consecutive order and following washes in PBS/Tween20, serially diluted serum samples, rec Protein-G-HRP and TMB SureBlueTM (SeraCare, MD) TMB Peroxidase Substrate were added to the plates. Optical density (OD) reads at 450 nm and 570 nm were performed using Biotek’s

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

M.S. Duthie et al. / Vaccine xxx (xxxx) xxx

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Fig. 1. Trial design schematic and profile. In A, the trial timeline is represented. The study comprised two periods: Period I (Study injection phase) that occurred from day 0 through the visit on day 84 and; Period II (Follow-up phase) that occurred after the day 84 visit and lasted through a final phone call visit on day 421. In B, a CONSORT diagram of screening, sequential enrolment into the two arms, follow-up, and analysis is shown.

Synergy2 plate reader (Biotek, VT). Endpoint titers were calculated using OD 450 nm 570 nm values for interpolating unknowns from the last value greater than the threshold given by naïve serum using a 4-parameter logistic model (Parameter 208) XL-Fit software as a Microsoft Excel add-in. Whole blood assays were conducted to evaluate antigenspecific cytokine secretion from reactive cells. Briefly, 900 ml blood was incubated with 100 ml of the following: 1X PBS (negative control) or 100 mg/ml LEP-F1 or 50 mg/ml PHA for 22 to 24 h at 37 °C. Post stimulation samples were each transferred to a Serum Separator (Sarstedt), centrifuged, supernatant collected and stored at 70 °C. until analysis. Cytokine content was measured by a quantitative multiplex bead array (Meso Scale Discovery [MSD]) to measure the level of each analyte (IFN-c, TNF, and IL-2). Specimens

from all visit days for a given subject and analyte were assayed on the same day, with each MSD plate containing standards and controls that were used to determine plate validity. Antigen-specific cytokine production was calculated as the concentration values obtained from the LEP-F1-stimulated samples minus the values obtained from negative control (PBS only added) samples, where the measured concentration was considered to be the average of duplicate wells. 2.5. Statistical methods No formal sample size calculations were performed for this Phase 1 safety trial; the analyses presented are exploratory. The trial was open-label, with all subjects receiving LepVax

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

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(LEP-F1 + GLA-SE). Data were assumed to be nonparameteric and were analyzed by the Mann Whitney test in GraphPad Prism software.

Table 2 Subjects with Adverse Events by Injection Interval, Severity, and Relatedness to Study Injection (Safety Population).

3. Results 3.1. Enrollment In all, 43 subjects were screened, of whom 19 (44%) were ineligible or not selected, and 24 (56%) were randomized and enrolled (Fig. 1B). One subject was screened twice: the first time for Treatment Group 1 for which they were not selected, and a second time for Treatment Group 2, for which they were randomized and enrolled. Sixteen (66.6%) of the enrolled subjects were granted Medical Monitor approval for certain laboratory eligibility criteria that were deemed not clinically significant. The demographic composition (age, gender, BMI) of each group was similar with no important differences among them (Table 1). Two subjects (8%) received one study injection and one subject (4%) received two study injections. Twenty-one subjects (88%) received all three injections (Fig. 1B).

Any adverse event Grade 1 Grade 2 Grade 3 Grade 4 Serious adverse event Injection Interval 1 Injection Interval 2 Injection Interval 3 Study injectionrelated Possibly Probably Definitely

2 lg LEP-F1 + 5 lg GLASE

10 lg LEP-F1 + 5 lg GLASE

12 (100%) 12 (100%) 2 (16.7%) 1 (8.3%) 0 0

12 (100%) 12 (100%) 1 (8.3%) 0 0 0

10 (83.3%) 10 (83.3%) 5 (41.7%) 10 (83.3%)

11 (91.7%) 10 (83.3%) 8 (66.7%) 11 (91.7%)

3 (25.0%) 3 (25.0%) 10 (83.3%)

3 (25.0%) 8 (66.7%) 11 (91.7%)

Table 3 Subjects with Adverse Events Occurring in > 5% Subjects (Safety Population).

3.2. Adverse events during the study injection period (period i; day 0– 84) During the study injection period (Period I; day 0–84), local and systemic reactogenicity was common with AEs reported by all (100%) of the study participants (Table 2). The highest grade reported for any one type of AE was as follows: Grade 1 in 100% and 100%; Grade 2 in 17% and 8%; and Grade 3 in 8% and 0% in Groups 1 and 2, respectively. No Grade 4 AEs, no SAEs, and no AESIs were reported during this period. Most subjects reported some injection site tenderness and/or pain (Table 3), although the frequency of this was lower after the third injection (Table 4). Local injection site reaction AEs were assessed 60 min after study injection administration at the study site, and documented in subject memory aids that were completed daily and reviewed on the second and seventh days after each study injection. The majority of local reactions were reported at the 2 day evaluation and most study injection-related AEs were of Grade 1 or Grade 2 severity, with one instance of Grade 3 injection site pain. Local study injection-related AEs were the most common AE, occurring in 83% of Group 1 and 92% of Group 2 subjects, respectively. Table 1 Summary of demographic data and other baseline characteristics.

Gender Female Male Race Black or African American White Age (yrs) Mean Std deviation Std error Minimum Median Maximum Body mass index (kg/m2) Mean Std deviation Std error Minimum Median Maximum

2 lg LEP-F1 + 5 lg GLASE

10 lg LEP-F1 + 5 lg GLASE

6 (50.0%) 6 (50.0%)

5 (41.7%) 7 (58.3%)

2 (16.7%)

1 (8.3%)

10 (83.3%)

11 (91.7%)

36.5 12.28 3.55 20 36.0 54

40.9 9.34 2.70 28 40.0 55

24.9 3.80 1.10 16 25.0 31

26.5 2.84 0.82 22 26.5 31

Injection related reactions Arthralgia Chills Decreased appetite Fatigue Headache Injection site pain Myalgia General AEs Dizziness Headache Laceration Oropharyngeal pain Rhinorrhoea Upper respiratory tract infection Laboratory parameters increased Blood alkaline phosphatase Blood potassium Blood pressure, diastolic Blood pressure, systolic

2 lg LEP-F1 + 5 lg GLA-SE

10 lg LEP-F1 + 5 lg GLA-SE

1 (8.3%) 2 (16.7%) 2 (16.7%) 5 (41.7%) 2 (16.7%) 10 (83.3%) 1 (8.3%)

1 (8.3%) 3 (25.0%) 1 (8.3%) 7 (58.3%) 3 (25.0%) 11 (91.7%) 4 (33.3%)

2 2 2 2 2 1

(16.7%) (16.7%) (16.7%) (16.7%) (16.7%) (8.3%)

0 1 0 3 1 2

2* (16.7%)

0

1 (8.3%) 1 (8.3%) 3 (25.0%)

1 (8.3%) 2 (16.7%) 0

(8.3%) (25.0%) (8.3%) 16.7%)

* One subject demonstrated an AE with elevations in both alkaline phosphatase and potassium levels.

Similar to local AEs, the majority of systemic reactions were reported at the 2 day evaluation timepoint and all reactions were Grade 1 or Grade 2. Solicited systemic reactions observed during the 7-day period following study injection occurred in 50% of Group 1 and 67% of Group 2 subjects, respectively. The most frequently reported solicited systemic reaction was fatigue (reported in 12 (50%) subjects), followed by chills, headache, and myalgia (Table 3). While all hematology assessments (hemoglobin, WBC count, platelet count) remained in the normal range, alkaline phosphatase and potassium levels were increased in a small number of individuals (2 total subjects and 1 subject in the 2 lg and 10 lg LEP-F1 groups, respectively) (Table 3). Given the small number of individuals and the limited range of altered parameters, these increases were not considered to be related to treatment. 3.3. Adverse events during the study post-injection follow-up period (Period II; day 85–421) During the follow-up period (day 85 to day 421), no AEs were reported among the subjects. No deaths occurred during the study.

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

M.S. Duthie et al. / Vaccine xxx (xxxx) xxx Table 4 Subjects with Solicited Local and Systemic Reaction Adverse Events by Post-Injection Period (Safety Population).

Any local reaction Post-injection Period 1 Post-injection Period 2 Post-injection Period 3 Bruising Erythema Haemorrhage Induration Pain Reaction Any systemic reaction Post-injection Period 1 Post-injection Period 2 Post-injection Period 3 Arthralgia Chills Decreased appetite Fatigue Headache Myalgia

2 lg LEP-F1 + 5 lg GLASE

10 lg LEP-F1 + 5 lg GLASE

10 (83.3%) 8 (66.7%)

11 (91.7%) 10 (83.3%)

9 (75.0%)

8 (66.7%)

5 (41.7%)

5 (41.7%)

0 0 1 (8.3%) 0 10 (83.3%) 0 6 (50.0%) 4 (33.3%)

1 (8.3%) 1 (8.3%) 0 1 (8.3%) 11 (91.7%) 1 (8.3%) 8 (66.7%) 8 (66.7%)

4 (33.3%)

4 (33.3%)

2 (16.7%)

0

1 2 2 5 2 1

1 3 1 7 3 4

5

the production of proinflammatory Th1-type cytokines increased following incubation of whole blood with LEP-F1 (Fig. 3 and Fig. S1). Relative to baseline samples, the amount of IFN-c, IL-2 and TNFa elicited by LEP-F1 was significantly increased at days 35 and 63 in each treatment group. 4. Discussion

(8.3%) (16.7%) (16.7%) (41.7%) (16.7%) (8.3%)

(8.3%) (25.0%) (8.3%) (58.3%) (25.0%) (33.3%)

3.4. LepVax-induced LEP-F1-specific immune responses Although antibodies are not essential within the immune profile associated with protection against M. leprae infection, we collected blood to assess serum anti-LEP-F1 antibody responses as an indication of effective immunization. Indeed, subjects vaccinated with LEP-F1 + GLA-SE at both dose levels demonstrated robust LEP-F1-specific IgG antibody titers (Fig. 2). Not surprisingly, the responses progressively increased after each injection, such that at day 35 the proportion of subjects showing at least a 4-fold increase in antibody response to LEP-F1 was 91% and 64% in the low and high dose antigen groups, respectively and at day 63, all subjects in both groups had attained levels indicative of antibody responsiveness. Although statistically significant differences were not observed, there was a trend toward higher overall IgG titers and response rates among subjects injected with LepVax containing the lower dose of 2 lg LEP-F1. Analysis of cellular responses induced by immunization with LEP-F1 + GLA-SE demonstrated that

The trial presented here is the first-in-human assessment of a defined vaccine candidate against leprosy. This represents an important milestone in the development of a vaccine against a human pathogen that cannot be grown in culture. AEs associated with LepVax were generally mild, transient, and typical of vaccines provided by intramuscular injection. No serious adverse events, adverse events of special interest, or Grade 4 adverse events occurred, and no subjects withdrew because of an AE. In addition, LepVax elicited robust production of circulating LEP-F1-specific IgG antibodies and generated LEP-F1-specific Th1 cells, as indicated by increased production of IFN-c, TNF, and IL-2 upon antigenic recall in whole blood samples. Thus, at both 2 lg and 10 lg doses of LEP-F1 protein, LepVax was safe, well tolerated and appropriately immunogenic when provided as three intramuscular injections at 28 day intervals to healthy adult subjects. Leprosy can be clinically cured with a 6–12 month multidrug therapy (MDT) regimen that has been distributed free of charge for registered leprosy patients in the drive toward ‘‘elimination” of leprosy as a global health problem. Since MDT’s introduction, global incidence has decreased, but detection rates have levelled off at around 220,000 new cases per year, a number that is generally believed to be a significant underestimate [3,21]. Transmission continues and the disease is re-emerging in many regions that previously reported elimination. Thus, leprosy remains a critical public health priority despite being targeted by the London Declaration on Neglected Tropical Diseases for elimination by 2020 [22]. Current strategies for preventing transmission rely on MDT for infected patients and post-exposure chemoprophylaxis of at-risk persons [23,24]. These have limitations in terms of sustainability and durability, however, and are unlikely to achieve eradication. Unlike drug treatment, vaccines could potentially provide active and sustained protection in both uninfected and infected individuals. Although an effective vaccine could be a critical element of control and may be essential for eradication, no effective leprosy vaccine exists and attempts to vaccinate humans have been limited. Meta–analyses of clinical trials of the BCG vaccine, which is administered predominantly with tuberculosis in mind, reveal a modest efficacy in preventing leprosy (0–41%) [16,17,25] and the

Fig. 2. Progressive increase in anti-LEP-F1 IgG antibody responses. Blood was collected and serum samples prepared from each subject at various times and were assessed for IgG antibodies against LEP-F1 by ELISA. Data are shown as individual points indicating the endpoint titer for each particular sample, with geometric mean titer and 95% confidence interval represented by the horizontal and vertical bars, respectively. **** = p-value < 0.001 comparing the indicated times by Mann Whitney test. No statistical differences (p-value > 0.05) were observed between individuals that received the either the 2 or 10 lg antigen doses on days 0, 35 and 63, repsectively.

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

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Fig. 3. LEP-F1 specific cytokine production over time. Blood was collected from each subject at various times and incubated with LEP-F1 protein or PBS alone (negative control). After 22–24 h plasma was collected and stored until cytokine content was assessed by multiplex MSD assay. Data are shown as individual points indicating the A. IFN-c B. TNF and C. IL-2 concentration within each particular sample, with geometric mean titer and 95% confidence interval represented by the horizontal and vertical bars, respectively. **, *** and **** = p-value < 0.01, < 0.005 and < 0.001, respectively, comparing the indicated times by Mann Whitney test. No statistical differences (p-value > 0.05) were observed between individuals that received the either the 2 or 10 lg antigen doses on days 0, 35 and 63, repsectively.

persistence of leprosy in regions with good BCG coverage indicates that additional strategies are required. ImmunvacÒ (formerly MIP, Mycobacterium indicus pranii) is licensed for adjunctive therapy for TB and similar evaluations for leprosy indicate that it leads to more rapid attainment of smear negativity compared to MDT alone, but reactional episodes have been observed [26–29]. The ideal vaccine would intercept M. leprae transmission and protect exposed individuals from developing disease. Demonstrating that LepVax is safe among individuals in a nonleprosy endemic region is a first step toward the implementation of an effective leprosy vaccine. Future evaluations in leprosyendemic regions will be performed to evaluate the safety and immune response profiles of LepVax in a blend of asymptomatic and symptomatic M. leprae-infected individuals. This is important because some concerns have been raised that eliciting anti-M. leprae immune responses among individuals already infected with the mycobacteria could be detrimental [30,31]. Indeed, immunization with the complete mycobacterial BCG vaccine elicits a

paucibacillary (PB) presentation in a small subset of individuals known to have elevated exposure to M. leprae infection through prolonged contact with patients [30,31]. In addition, estimates are that 5–7 years elapse between primary M. leprae infection and the emergence of symptoms. It is therefore not surprising that leprosy most commonly manifests in adults. M. leprae infection could well become established in childhood, however, and it will be important to evaluate LepVax in adolescents and children who could potentially become infected. There is strong associative evidence that the genetic background and immune response influence both susceptibility to M. leprae infection and outcome of leprosy [32–35]. Although IFNcproducing CD4 T cells are required for control of M. leprae replication in the mouse footpad model, induction of such Th1 responses is not entirely sufficient to protect against disease [36]. Indeed, both presumably protected healthy household contacts and symptomatic PB leprosy patients display antigen-specific IFNc responses. Several studies have demonstrated that CD4 T cells that

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

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secrete only IFN-c have a limited capacity to develop into memory cells, but that the proportion of multifunctional Th1 cells (characterized by their simultaneous secretion of multiple cytokines [IFN-c, IL-2 and TNF]) positively correlates with protection against various cancers and infectious diseases [37–40]. Our recent multiparameter flow cytometry analyses revealed that the population of multifunctional ML2028-specific T cells observed in healthy household contacts was larger than that observed in PB patients and suggests that these multifunctional antigen-specific T cells provide a more effective response against M. leprae infection that prevents the development of leprosy [41]. The importance of the simultaneous secretion of different Th1 cytokine combinations, and therefore the memory differentiation and functionality of antigen-specific CD4 T cells, for protective immunity can only be formally demonstrated in an efficacy trial. Escalation of the LEP-F1 dose from 2 lg and 10 lg in LepVax did not alter the kinetics of, nor did it result in greater, antigen-specific antibody or cytokine responses. These data suggest that the lower antigen dose might be suitable for subsequent efficacy studies of the vaccine candidate, and use of the lower dose would be preferable for future implementation as more doses would be available. Several trials have observed peaked dose–response curves for vaccines where antigen-specific T cell responses induced by the highest provided dose are not superior, and sometimes lower, than those induced by lower vaccine doses [42–45]. It is suggested that high doses can push T cells to an exhausted state with reduced functional avidity and increased differentiation into a terminal state [46,47]. Further analyses of antigen (such as crude M. leprae and/or single protein)-specific T cell responses, including investigation of both CD4 and CD8 T cell responses by flow cytometry are required to establish if the differing LepVax doses induced truly comparable functional attributes among antigen-specific T cells. Making sustained impact for global health will only occur if several requirements are met, including suitable safety, efficacy, supply, consistency, and cost. As a subunit vaccine LepVax can be produced at sufficient scale and at a sufficiently low cost to provide a sustainable vaccine solution. Future efficacy trials will be performed to determine whether LepVax can provide protection from clinical disease and if LepVax can become a component of a multimodal strategy to control leprosy. For efficiency in both recruiting and observational periods, efficacy trials would ideally be conducted among those known to be at highest risk of developing leprosy, that is, residents of highly endemic areas who are also household contacts of confirmed, registered multibacillary cases. The emergence of a prophylactic defined subunit vaccine against leprosy would represent a major breakthrough. The encouraging safety and immunogenicity data reported here provide a strong rationale for further testing of the LepVax vaccine and advancement to efficacy trials in leprosy endemic countries.

CRediT authorship contribution statement Malcolm S. Duthie: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft, Writing - review & editing. Aude Frevol: Methodology, Project administration, Supervision. Tracey Day: Formal analysis, Investigation, Supervision, Writing - original draft. Rhea N. Coler: Conceptualization, Investigation, Methodology, Supervision, Writing - review & editing. Julie Vergara: Formal analysis, Investigation, Methodology. Tom Rolf: Investigation, Methodology. Zachary K. Sagawa: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing original draft, Writing - review & editing. Anna Marie Beckmann: Methodology, Project administration, Supervision. Corey Casper:

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Supervision. Steven G. Reed: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was conducted with grant funding from the American Leprosy Missions, with important contributions from Action Damien, Deutsche Lepra- und Tuberkulosehilfe e.V., effect: hope, Fairmed, Fondation Raoul Follereau, Netherlands Leprosy Relief, and Secours aux Lepreuz – Leprosy Relief Canada. The authors wish to thank the personnel at Covance Clinical Research Unit (Madison, WI), Medical Monitor Dr. Stuart Kahn, and the study volunteers. The development of LepVax and its progress to the Phase 1 trial described here would not have been achieved without invaluable insight provided by a plethora of leprosy researchers and clinicians, in particular Drs. Patrick Brennan, Warwick Britton, Tom Gillis, Christa Kasang, and Paul Saunderson. The funders had no input to the production of this manuscript and the opinions provided are the authors0 own. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.12.050. References [1] Scollard DM, Truman RW, Ebenezer GJ. Mechanisms of nerve injury in leprosy. Clin Dermatol 2015;33(1):46–54. [2] Wilder-Smith EP, Van Brakel WH. Nerve damage in leprosy and its management. Nat Clin Pract Neurol 2008;4(12):656–63. [3] Smith WC, van Brakel W, Gillis T, Saunderson P, Richardus JH. The missing millions: a threat to the elimination of leprosy. PLoS Negl Trop Dis 2015;9(4): e0003658. [4] Global leprosy update. 2016: accelerating reduction of disease burden. Wkly Epidemiol Rec 2017;92(35):501–19. [5] Barreto JG, Guimaraes Lde S, Frade MA, Rosa PS, Salgado CG. High rates of undiagnosed leprosy and subclinical infection amongst school children in the Amazon region. Mem Inst Oswaldo Cruz 2012;107(Suppl 1):60–7. [6] de Souza MM, Netto EM, Nakatani M, Duthie MS. Utility of recombinant proteins LID-1 and PADL in screening for Mycobacterium leprae infection and leprosy. Trans R Soc Trop Med Hyg 2014;108(8):495–501. [7] Frade MA, de Paula NA, Gomes CM, Vernal S, Bernardes Filho F, Lugao HB, et al. Unexpectedly high leprosy seroprevalence detected using a random surveillance strategy in midwestern Brazil: a comparison of ELISA and a rapid diagnostic test. PLoS Negl Trop Dis 2017;11(2):e0005375. [8] Zodpey SP, Bansod BS, Shrikhande SN, Maldhure BR, Kulkarni SW. Protective effect of bacillus calmette guerin (BCG) against leprosy: a population-based case-control study in Nagpur India. Lepr Rev 1999;70(3):287–94. [9] Zodpey SP, Ambadekar NN, Thakur A. Effectiveness of bacillus calmette guerin (BCG) vaccination in the prevention of leprosy: a population-based casecontrol study in Yavatmal District India. Publ Health 2005;119(3):209–16. [10] Rodrigues LC, Kerr-Pontes LR, Frietas MV, Barreto ML. Long lasting BCG protection against leprosy. Vaccine 2007;25(39–40):6842–4. [11] WHO. Global tuberculosis programme and global programme on vaccines. Statement on BCG revaccination for the prevention of tuberculosis. Wkly Epidemiol Rec 1995;70(32):229–31. [12] Rodrigues LC, Pereira SM, Cunha SS, Genser B, Ichihara MY, de Brito SC, et al. Effect of BCG revaccination on incidence of tuberculosis in school-aged children in Brazil: the BCG-REVAC cluster-randomised trial. Lancet 2005;366 (9493):1290–5. [13] Barreto ML, Pereira SM, Ferreira AA. BCG vaccine: efficacy and indications for vaccination and revaccination. J Pediatr (Rio J) 2006;82(3 Suppl):S45–54. [14] Convit J, Sampson C, Zuniga M, Smith PG, Plata J, Silva J, et al. Immunoprophylactic trial with combined Mycobacterium leprae/BCG vaccine against leprosy: preliminary results. Lancet 1992;339(8791):446–50.

Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050

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Please cite this article as: M. S. Duthie, A. Frevol, T. Day et al., A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA–SE) in healthy adults, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.050