An open-label, Phase 3 trial of TAK-003, a live attenuated dengue tetravalent vaccine, in healthy US adults: immunogenicity and safety when administered during the second half of a 24-month shelf-life

ABSTRACT

Dengue is caused by a mosquito-transmitted flavivirus. The disease is now endemic to many tropical and subtropical regions, manifesting as approximately 96 million symptomatic cases of dengue each year. Clinical trials have shown TAK-003 (Qdenga®), a live attenuated dengue tetravalent vaccine, to be well-tolerated, immunogenic, and efficacious in adults with no prior exposure to dengue virus infection living in non-endemic regions, as well as in adults and children living in dengue-endemic areas. This open-label, single-arm phase 3 trial (NCT03771963) was conducted in two dengue non-endemic areas of the USA, and it evaluated the immunogenicity and safety of naturally-aged TAK-003 administered to adult participants. Overall, the immunogenicity data from this trial are consistent with those reported from other TAK-003 phase 2 and 3 trials, and the safety data are consistent with the broader integrated safety data analysis. The data show that naturally-aged TAK-003 had a well-tolerated reactogenicity and adverse events profile when administered in the second half of its clinical 24-month shelf-life and that it still elicited an immune response that persisted up to 6 months after the second dose against all four dengue serotypes, with no important safety risks identified during the trial.

KEYWORDS:

• Dengue fever
• immunogenicity
• live attenuated
• safety
• shelf-life
• tetravalent
• dengue vaccine

Introduction

Dengue is caused by infection with the dengue virus (DENV), a flavivirus with a ribonucleic acid genome that occurs as four recognized DENV serotypes, DENV-1, DENV-2, DENV-3, or DENV-4.¹ The virus is transmitted to humans via bites from virus-infected Aedes aegypti or Aedes albopictus mosquitoes. Approximately 96 million cases of dengue manifest clinically every year, with 500,000 requiring hospitalization.^1,2 Illness owing to infection by any of the four co-circulating DENV serotypes in endemic regions can result in a range of symptoms, from asymptomatic disease to debilitating but transient dengue fever, life-threatening dengue hemorrhagic fever, and dengue shock syndrome.^3–5

Infection with one DENV serotype produces life-long homologous immunity but may increase the risk of severe disease following a secondary heterotypic infection:^6,7 81% of DENV-diagnosed children in one study presented with a secondary DENV infection and, of these, 53% had dengue hemorrhagic fever.⁷ Vaccine development has, therefore, focused on tetravalent vaccines that generate protective responses against the four serotypes.^8,9 Trials with an alternative licensed dengue vaccine, CYD-TDV (Dengvaxia®), revealed a high CYD-TDV efficacy for baseline seropositive recipients at the time of vaccination, but a much lower efficacy in those who were baseline seronegative.^10,11 Moreover, the reduced efficacy for seronegative recipients disproportionately affected young-aged children. Owing to CYD-TDV vaccination increasing the risk of hospitalization in baseline seronegative individuals, it is licensed for administration only to baseline dengue seropositive individuals.¹² Consequently, two laboratory tests are required to confirm prior dengue exposure before CYD-TDV is administered as three doses 6-months apart (i.e., at months 0, 6, and 12).¹³ This presents an arduous challenge both in terms of accomplishing the laboratory tests and the potential for incomplete dosing compliance before full protection is conferred. There is, therefore, a significant unmet clinical need for an effective dengue vaccine that can be used to protect both seronegative and seropositive individuals who are at risk from DENV infection.¹⁴

TAK-003 (Qdenga®) is a live attenuated dengue tetravalent vaccine that comprises four DENV strains: an attenuated dengue serotype 2 strain (DENV-2) and three recombinant strains comprising the DENV-2 backbone and the respective pre-membrane and envelope genes of DENV-1, DENV-3, and DENV-4 serotypes² (Figure 1). Phase 1 and Phase 2 clinical trials have shown TAK-003 to be well tolerated and immunogenic in healthy adults with no prior exposure to DENV infection living in dengue non-endemic regions as well as in adults and children living in dengue-endemic areas such as Asia and Latin America.^15–18 Furthermore, TAK-003 is efficacious in preventing dengue in children and adolescents in endemic countries.^19,20 To date, TAK-003 has been licensed for prevention against dengue in the European Union, Great Britain, Brazil, Argentina, Thailand, and Indonesia, among other countries.²¹

Figure 1. Genetic structure of the four TAK-003 vaccine strains. Abbreviations: C, capsid; E, envelope; NS, nonstructural protein; prM, premembrane; TDV-1/2/3/4, dengue serotype 1/2/3/4 strain. Reproduced with permission from Patel et al.⁴².

This open-label, single-arm phase 3 trial (DEN-307; NCT03771963) evaluated the immunogenicity and safety of naturally-aged TAK-003, administered as two doses 3 months apart, after storage at 2°C to 8°C for 14–20 months (i.e., more than half of its clinical 24-month shelf-life). The objective for this trial was to quantify the neutralizing antibody response that is elicited following the administration of TAK-003 during the latter half of its 2-year shelf-life.

Materials and methods

Trial design

A single trial group of 200 healthy adult participants was enrolled at two centers (Anaheim, CA, and Hutchinson, KS) in the USA. Participants received a subcutaneous injection of naturally-aged (14–20 months in storage at 2°C to 8°C) TAK-003 on Day 1 and on Day 90 (Figure 2). Trial duration was approximately 270 days (9 months) for each participant. The first trial participant received the first TAK-003 dose on 28 March 2019 and the last participant received the second TAK-003 dose on 9 September 2019, corresponding to 14–20 months of a single batch of TAK-003 vaccine aging during storage at 2°C to 8°C.

Figure 2. Trial visits schematic. Abbreviations: MAAEs, medically attended adverse events; SAEs, serious adverse events. Safety assessments were as follows: solicited local events (recorded up to 30 minutes, and up to 7 days after each TAK-003 dose), solicited systemic events (recorded up to 30 minutes, and up to 14 days after each TAK-003 dose), unsolicited events (recorded up to 28 days after each TAK-003 dose), and MAAEs and SAEs recorded for the entire duration of trial. Solicited local AEs comprised injection site pain, injection site erythema, and injection site swelling. Solicited systemic AEs comprised asthenia, headache, malaise, myalgia, and fever. MAAEs were defined as AEs leading to an unscheduled visit to, or by, a healthcare professional including visits to an emergency department, but not fulfilling seriousness criteria.

Participants

Eligible participants were enrolled if they were 18–60 years old at the time of entry into the trial, male or female, and in good health as determined by medical history, physical examination (including vital signs), and the clinical judgment of the investigator, and, if they could comply with trial procedures for the duration of the trial. The trial design did not stratify participant enrollment for any given variable; all participants were enrolled over a 7-month period as per study eligibility criteria.

The trial excluded participation from individuals with hypersensitivity to any TAK-003 formulation component, impaired or altered immune function, planned or ongoing pregnancy or breastfeeding, or previous vaccination against dengue fever. Detailed exclusion criteria are provided in Table S1.

Any participant was eligible to enroll voluntarily in this trial without prejudice to their baseline dengue serostatus. Baseline serostatus for each participant was ascertained subsequently from a blood sample obtained immediately prior to the first TAK-003 dose.

Participants for this trial were solicited from a database/register at each respective site and supplemented by recruitment via Independent Review Board (IRB)-approved advertisements on social media. Written informed consent was obtained from each participant before enrollment; this IRB-approved trial was conducted in accordance with the Declaration of Helsinki and the International Council for Harmonization tripartite guidelines for good clinical practice.^22,23 IRB oversight for this study (tracking number: 20190261) was conducted by the Copernicus Group Independent Review Board, 5000 Centre Green Way Suite 200, Cary, NC 27513, USA.

Immunogenicity evaluation

Immunogenicity assessments comprised the measurement of dengue neutralizing antibodies by microneutralization test 50% (MNT₅₀) for each of the four dengue serotypes using blood samples collected pre-vaccination on Day 1, and, at 1 month (Day 120) and 6 months (Day 270) after the second TAK-003 dose.

Safety evaluation

Solicited local adverse events (AEs), comprising injection site pain, injection site erythema, and injection site swelling, and, solicited systemic AEs comprising fever, headache, asthenia, malaise, and myalgia, were assessed in the clinic for 30 min immediately after each TAK-003 dose. Solicited local and systemic AEs were further recorded for 7 days (day of vaccination +6 days) and 14 days (day of vaccination +13 days), respectively, following each TAK-003 dose. Diary cards were distributed to all participants to record the solicited local and systemic AEs.

Unsolicited AEs assessed for 28 days (day of vaccination +27 days) following each TAK-003 dose were recorded for all participants. Serious AEs (SAEs), medically attended AEs (MAAEs), AEs leading to withdrawal from a second dose of TAK-003, and discontinuation from trial were documented for the entire trial duration. MAAEs were defined as AEs leading to an unscheduled visit to, or by, a health-care professional, including visits to an emergency department but not fulfilling seriousness criteria.

The severity unsolicited AEs was categorized by investigators as mild, moderate, or severe, based on trial protocol definitions (Table S2). In the instances where severity was not recorded for an unsolicited AE, it was imputed as severe for the purposes of data analysis. All solicited local AEs were considered related to TAK-003 administration. The investigators assessed, where applicable, the causal relationship between TAK-003 vaccine/trial procedures and solicited systemic AEs and/or unsolicited AEs; attribution to TAK-003 was binary: either it was related or not related, and both were defined as follows:

• Related: there is suspicion that there is a relationship between the trial vaccine and the AE (without determining the extent of probability); there is a reasonable possibility that the trial vaccine contributed to the AE.

• Not Related: there is no suspicion that there is a relationship between the trial vaccine and the AE; there are other more likely causes, and the administration of the trial vaccine is not suspected to have contributed to the AE.

Trial objectives

The primary trial objective was to describe, at 1 month (day 120) post-second dose, the neutralizing antibody response against each dengue serotype following administration of a naturally-aged batch of TAK-003.

Secondary objectives were to describe the seroconversion rates, for all dengue serotypes, at 1 month and at 6 months (day 270) post-second dose, and to also describe the neutralizing antibody response for each dengue serotypes at 9 months (Day 270). Seropositive status was defined as a reciprocal neutralizing titer ≥10 against at least one dengue virus serotype at baseline.

TAK-003 tolerability was evaluated following each vaccination dose by assessing solicited local AEs, solicited systemic AEs, unsolicited AEs, MAAEs, and SAEs.

Statistics

Descriptive statistics, including 95% confidence intervals (CIs), were provided for the primary and secondary immunogenicity endpoints; safety set, full analysis set (FAS), and per-protocol set (PPS) were generated. Safety Set comprised all enrolled participants who received at least one dose of TAK-003 (Figure 3a). FAS comprised all enrolled participants who received at least one dose of TAK-003 and for whom a valid pre-dose (baseline) measurement and at least one valid post-dose measurement were available for immunogenicity assessments. PPS comprised all participants from the FAS who had no major protocol violations, but excluding participants who were baseline seropositive for any serotype of dengue virus at baseline.

Figure 3. (a) Participant allocation to safety, full analysis and per-protocol sets. *Any participant not included in the per-protocol set if he/she experienced one or more trial protocol deviation(s) and (b) Participant disposition for the DEN-307 clinical trial. Abbreviations: AE, adverse event; LTF, lost to follow up; WOC, withdrawal of consent.

Results

Trial population and baseline characteristics

A total 200 participants were enrolled in this trial; of whom, 176 (88.0%) received two doses of TAK-003 (Figure 3b). Twenty-four (12.0%) participants did not receive their second TAK-003 dose: the majority either being lost to follow-up or withdrawing consent. The individual reasons for consent withdrawal were personal, and none was medically related.

A total 168 (84.0%) participants completed the trial, with 32 (16.0%) participants discontinued from the trial owing to them being lost to follow-up (n = 22) or withdrawing consent (n = 10).

Participant baseline demographic characteristics were generally consistent across trial analysis groups (Table 1). On average, the participants were 40–41 years old, approximately half were female participants, the majority (>70%) were white, and their mean BMI was approximately 27.5 kg/m². Some 14.1% of participants were dengue seropositive at baseline (safety set).

Table 1. Participant demographic and baseline characteristics.

	Safety set (N = 200)	Full analysis set (N = 174)	Per-protocol set (N = 139)
Mean age^a (SD), years	40.3 (12.0)	40.7 (12.0)	41.1 (11.7)
Female, n (%)	101 (50.5)	94 (54.0)	78 (56.1)
Hispanic or Latino, n (%)	64 (32.0)	58 (33.3)	45 (32.4)
Race, n (%)
White	148 (74.0)	133 (76.4)	102 (73.4)
Black or African American	41 (20.5)	32 (18.4)	29 (2.9)
Asian	5 (2.5)	4 (2.3)	4 (20.9)
Hawaiian or other Pacific Islander	1 (0.5)	1 (0.6)	1 (0.7)
Multiracial	3 (1.5)	2 (1.1)	1 (0.7)
Not reported	2 (1.0)	2 (1.1)	2 (1.4)
Mean BMI (SD), kg/m^2	27.5 (4.3)	27.6 (4.3)	27.5 (4.2)
Baseline dengue serostatus, n (%)
Seropositive	28 (14.1)	22 (12.6)	0
Seronegative	171 (85.9)	152 (87.4)	139 (100.0)

^aage range for all three analysis sets: 18.0 to 60.0 years.

Abbreviations: BMI, body mass index; n, number of participants; SD, standard deviation.

Immunogenicity

Seroconversion at Day 120 was evident for >97% of participants for all dengue serotypes (Figure 4a). By Day 270, there were ~12% fewer DENV-3 and ~13% fewer DENV-4 seropositive participants, whereas for DENV-1 and DENV-2 it remained relatively unchanged (Figure 4a).

Figure 4. (a) Percentage (95% CI) of participants who were seropositive against each DENV serotype at days 120 and 270 and (b) Percentage (95% CI) of trivalent and tetravalent baseline seropositive participants (per-protocol set). Baseline seropositive defined as a reciprocal neutralizing antibody titer ≥ 10 to $\ge$ 1 dengue virus serotypes. Baseline seronegative defined as titer < 10 to all dengue serotypes. Abbreviations: CI, confidence interval; DENV, dengue virus.

Trivalent seroconversion was observed in 99.2% of participants, and tetravalent seroconversion was seen in 97.0% of participants at Day 120 (Figure 4b). By Day 270, trivalent and tetravalent seroconversion rates had decreased to 91.0% and 78.9%, respectively (Figure 4b).

All participants included in PPS were seronegative for dengue at baseline. TAK-003 elicited dengue neutralizing antibody responses, as described by geometric mean titers (GMTs), against each of the four dengue serotypes at Day 120 (Figure 5). For each serotype, GMTs at Day 120 were in the following order from greatest to least: DENV-2, DENV-1, DENV-3, and DENV-4.

Figure 5. Geometric mean titers (95% confidence interval) of neutralizing antibodies measured by MNT₅₀ for each dengue serotype (per-protocol set). Abbreviations: DENV, dengue virus; MNT₅₀, microneutralization test 50%.

The immune response elicited following two doses of TAK-003 was persistent though somewhat attenuated (Figure 5). Between Day 120 and Day 270, GMTs decreased by 44.1%, 42.8%, 52.2%, and 51.8% for DENV-1, DENV-2, DENV-3, and DENV-4, respectively, and, at Day 270 remained highest for DENV-2 (1170.6) and lowest for DENV-4 (57.6).

Safety

Solicited local adverse events

Within the 30 min after the first TAK-003 dose, two (1.0%) participants experienced mild injection site pain, and, following the second dose, five (2.8%) participants experienced two events of mild pain, two events of mild erythema, and one event of mild swelling (Table 2).

Table 2. TAK-003 overview of adverse events.

	Any Vaccination (N = 200)		First vaccination (N = 200)		Second vaccination (N = 176)
	Events	Participants (%)	Events	Participants (%)	Events	Participants (%)
Any Solicited Adverse Events	454	89 (46.1)	279	69 (35.9)	175	54 (32.3)
30-min post TAK-003 dose	8	7 (3.5)	3	2 (1.0)	5	5 (2.8)
Local (within 7 days)	454	89/193 (46.1)	279	69/192 (35.9)	175	54/167 (32.3)
Systemic (within 14 days)	950	95/193 (49.2)	587	81/192 (42.2)	363	57/167 (34.1)
Related to TAK-003	893	94/193 (48.7)	557	79/192 (41.1)	336	55/167 (32.9)
Prolonged solicited local AEs (>7 days beyond diary recorded period)	15	10 (5.0)	12	8 (4.0)	3	2 (1.1)
Prolonged solicited systemic AEs (>14 days beyond diary recorded period)	4	3 (1.5)	1	1 (0.5)	3	2 (1.1)
Any Unsolicited AEs (within 28 days)^a	59	38 (19.0)	36	23 (11.5)	23	19 (10.8)
Related to TAK-003	16	11 (5.5)	14	9 (4.5)	2	2 (1.1)
Related to trial procedure	6	5 (2.5)	5	4 (2.0)	1	1 (0.6)
Mild	50	31 (15.5)	30	19 (9.5)	20	16 (9.1)
Moderate	9	7 (3.5)	6	4 (2.0)	3	3 (1.7)
Any Medically attended AEs^b	146	52 (26.0)	60	29 (14.5)	86	35 (19.9)
Related to TAK-003	2	1 (0.5)	2	1 (0.5)	0	0
Related to trial procedure	0	0	0	0	0	0
Mild	123	33 (16.5)	54	24 (12.0)	69	19 (10.8)
Moderate	23	19 (9.5)	6	5 (2.5)	17	16 (9.1)
Leading to vaccine withdrawal	5	5 (2.5)	5	5 (2.5)	0	0
Leading to discontinuation from trial	1	1 (0.5)	1	1 (0.5)	0	0
Any Serious Adverse Events^c	5	5 (2.5)	2	2 (1.0)	3	3 (1.7)
Moderate	4	4 (2.0)	1	1 (0.5)	3	3 (1.7)
Severe	1	1 (0.5)	1	1 (0.5)	0	0
Leading to vaccine withdrawal	1	1 (0.5)	1	1 (0.5)	0	0

^aNo reported unsolicited AE was severe, led to vaccine withdrawal or discontinuation from trial. ^bNo reported MAAE was severe. ^cNo reported serious AE was mild, related to vaccine or trial procedure, or led to discontinuation from the trial.

Any participant who experienced >1 adverse event is counted under each vaccine dose column, but is counted only once in the “Any vaccination” column.

Abbreviations: AE, adverse event; MAAE, medically attended adverse event; min, minutes; SAE, serious adverse event. Medically attended AEs were defined as AEs leading to an unscheduled visit to, or by, a healthcare professional, including visits to an emergency department, but not fulfilling seriousness criteria. Any MAAEs and SAEs were recorded throughout the trial duration.

Solicited local AEs within 7 days were reported by 69 (35.9%) participants after the first vaccination and by 54 (32.3%) after the second vaccination (Table 3). The majority of these events were of mild intensity with only one (0.5%) participant with severe injection site pain after the first dose and two (1.2%) others who also reported it as severe after the second dose (Table 3). Overall, injection site pain was the most frequently reported solicited local AE after either vaccine dose, followed by injection site erythema (Table 3).

Table 3. Summary of solicited local adverse events within 7 days post vaccination (safety set).

	Number (%) of participants
Adverse events	Any Vaccination (N = 200)	First Vaccination (N = 200)	Second Vaccination (N = 176)
Any solicited local adverse events	89 (46.1)	69 (35.9)	54 (32.3)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	74 (38.3)	59 (30.7)	48 (28.7)
Moderate	12 (6.2)	9 (4.7)	4 (2.4)
Severe	3 (1.6)	1 (0.5)	2 (1.2)
Pain
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Any event	80 (41.5)	57 (29.7)	52 (31.1)
Mild	67 (34.7)	49 (25.5)	46 (27.5)
Moderate	10 (5.2)	7 (3.6)	4 (2.4)
Severe	3 (1.6)	1 (0.5)	2 (1.2)
Erythema^a
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Any event	29 (15.0)	25 (13.0)	10 (6.0)
Mild: ≥2.5-≤5 cm	27 (14.0)	23 (12.0)	10 (6.0)
Moderate: >5-≤10 cm	2 (1.0)	2 (1.0)	0
Swelling^b
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Any event	9 (4.7)	8 (4.2)	4 (2.4)
Mild: ≥2.5-≤5 cm	9 (4.7)	8 (4.2)	4 (2.4)

^aNo reported severe erythema events. ^bNo reported moderate or severe swelling events.

Any participant who experienced >1 adverse event is counted under each vaccine dose column, but is counted only once in the “Any vaccination” column.

All AEs were considered attributable to TAK-003 by default. Severity of each event was attributable according to pre-defined trial protocol criteria (see Table S2).

Pain, erythema, and swelling events were to be diary-recorded by each participant up to 7 days after each TAK-003 dose.

Most solicited local AEs occurred within the first 3 days after vaccination and the most common day of onset of pain after any vaccination was on the day of vaccination, whereas erythema and swelling were evident on the next day after vaccination. The mean duration for local AEs after any vaccination ranged between 3 days (pain) and 3.6 days (swelling).

Prolonged solicited local AEs (that is, those that persisted beyond the 7-day diary period) were observed for eight (4.0%) and two (1.1%) participants after the first and second vaccination, respectively (Table 2); all events subsequently resolved within 14 days.

Solicited systemic adverse events

Participants experienced fewer solicited systemic AEs after the second TAK-003 dose (34.1%) than after the first dose (42.4%), and correspondingly fewer AEs after the second dose were considered attributable to the vaccine (Table 2).

Headache was the most frequently reported event followed by myalgia, malaise, and asthenia (Table 4). No participant experienced any fever ≥39°C (Table 4).

Table 4. Summary of solicited systemic adverse events within 14 days post vaccination (safety set).

	Number (%) of participants
Adverse events	Any Vaccination (N = 200)	First Vaccination (N = 200)	Second Vaccination (N = 176)
Any solicited systemic adverse events	95 (49.2)	81 (42.2)	57 (34.1)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	64 (33.2)	59 (30.7)	43 (25.7)
Moderate	25 (13.0)	17 (8.9)	11 (6.6)
Severe	6 (3.1)	5 (2.6)	3 (1.8)
Related to TAK-003	94 (48.7)	79 (41.1)	55 (32.9)
Headache (any event)	70 (36.3)	58 (30.2)	38 (22.8)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	47 (24.4)	44 (22.9)	27 (16.2)
Moderate	19 (9.8)	11 (5.7)	9 (5.4)
Severe	4 (2.1)	3 (1.6)	2 (1.2)
Related to TAK-003	67 (34.7)	55 (28.6)	36 (21.6)
Myalgia (any event)	64 (33.2)	49 (25.5)	37 (22.2)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	53 (27.5)	40 (20.8)	34 (20.4)
Moderate	8 (4.1)	6 (3.1)	2 (1.2)
Severe	3 (1.6)	3 (1.6)	1 (0.6)
Related to TAK-003	64 (33.2)	48 (25)	37 (22.2)
Asthenia (any event)	39 (20.2)	32 (16.7)	20 (12.0)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	23 (11.9)	20 (10.4)	13 (7.8)
Moderate	12 (6.2)	10 (5.2)	5 (3.0)
Severe	4 (2.1)	2 (1.0)	2 (1.2)
Related to TAK-003	37 (19.2)	31 (16.1)	19 (11.4)
Malaise (any event)	48 (24.9)	38 (19.8)	22 (13.2)
N evaluated	193 (96.5)	192 (96.0)	167 (94.9)
Mild	31 (16.1)	25 (13.0)	15 (9.0)
Moderate	12 (6.2)	9 (4.7)	5 (3.0)
Severe	5 (2.6)	4 (2.1)	2 (1.2)
Related to TAK-003	48 (24.9)	37 (19.3)	22 (13.2)
Fever^a (any event)	6 (3.1)	5 (2.6)	2 (1.2)
N evaluated	191 (95.5)	189 (94.5)	166 (94.3)
38-<38.5°C	4 (2.1)	4 (2.1)	1 (0.6)
38.5-<39°C	2 (1.0)	1 (0.5)	1 (0.6)
Related to TAK-003	6 (3.1)	5 (2.6)	1 (0.6)

^aNo reported events of fever ≥39°C.

Any participant who experienced >1 adverse event is counted under each vaccine dose column, but is counted only once in the “Any vaccination” column.

Severity of AE assessed by participant, and attributability to TAK-003 assessed by the investigator.

All five solicited adverse events were to be diary-recorded by each participant up to 14 days after each TAK-003 dose.

Most of the solicited systemic events experienced were either mild or moderate after either dose of TAK-003: five (2.6%) participants experienced severe AEs after the first dose and three (1.8%) after the second dose (Table 4).

The majority of solicited systemic AEs occurred within the first 7 days after vaccination; the most common day of onset for headache, myalgia, malaise, and asthenia was on the day of vaccination. The mean duration of solicited systemic AEs after vaccination ranged between 3.0 days (myalgia) and 4.2 days (fever).

Prolonged solicited systemic AEs (that is, those that persisted beyond the 14-day diary period) were reported by one (0.5%) participant after the first vaccination, and two (1.1%) participants after the second vaccination (Table 2); all events subsequently resolved within 20 days.

Unsolicited AEs

Thirty-eight (19.0%) participants experienced a total 59 unsolicited events, none of which was severe; 50 (84.7%) events were mild and 9 (15.3%) were moderate in intensity (Table 2).

A total 16 events reported by 11 participants (5.5%) were considered attributable to TAK-003 administration, with the majority (14 events in 9 participants) reported after the first dose (Table 2). All events considered attributable to TAK-003 after the first dose were: ocular hyperemia, abdominal discomfort, diarrhea, injection site events (bruising, pain, pruritus, induration), pain, headache, pharyngeal pain, pruritus, and rash. Those considered attributable to TAK-003 after the second dose were injection bruising and injection site induration. The majority of TAK-003 related unsolicited AEs were mild in intensity, and no event was rated as severe.

The most frequently reported unsolicited AEs, by preferred term (PT), were abdominal discomfort and upper respiratory tract infection, each reported in three (1.5%) participants in total (Table 5). There were no individual unsolicited AEs, by PT, that were reported by >4 (>2.0%) participants. Other AEs reported within 28 days of dose were singly occurring events, all of which were of mild or moderate intensity. All unsolicited AEs resolved, and none led to withdrawal from administration of the second TAK-003 dose or to participant discontinuation from the trial.

Table 5. Summary of unsolicited adverse events up to 28 days post vaccination, by preferred term (safety set).

	Any vaccination (N = 200)		First vaccination (N = 200)		Second vaccination (N = 176)
Adverse events	Events	Participants (%)	Events	Participants (%)	Events	Participants (%)
Any unsolicited adverse event	59	38 (19.0)	36	23 (11.5)	23	19 (10.8)
Unsolicited adverse events experienced by ≥2 participants, by preferred term
Abdominal discomfort	3	3 (1.5)	2	2 (1.0)	1	1 (0.6)
Upper respiratory tract infection	3	3 (1.5)	2	2 (1.0)	1	1 (0.6)
Dyspepsia	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Injection site bruising	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Injection site induration	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Injection site pain	2	2 (1.0)	2	2 (1.0)	0	0
Injection site pruritus	2	2 (1.0)	2	2 (1.0)	0	0
Arthropod bite	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Oropharyngeal pain	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Sleep apnea syndrome	2	2 (1.0)	2	2 (1.0)	0	0

Any participant who experienced >1 adverse event is counted under each vaccine dose column, but is counted only once in the “Any vaccination” column.

All unsolicited adverse events were evaluated up to 28 days after each TAK-003 dose.

Medically attended AEs

MAAEs (146 events in total) after any vaccination were experienced by 52 (26.0%) participants: all events were either mild or moderate in intensity (Table 2). The most common MAAEs, by PT, were acute sinusitis (4.5% of participants), bronchitis (2.0%), and dermatitis contact (2.0%), and they were the only ones reported with a frequency of ≥2% (Table 6). Other MAAEs reported were singly occurring events, none of which was a severe event. All MAAEs resolved.

Table 6. Summary of medically attended adverse events (MAAEs) post vaccination up to the end of study (safety set).

	Any vaccination (N = 200)		First vaccination (N = 200)		Second vaccination (N = 176)
Adverse events	Events	Participants (%)	Events	Participants (%)	Events	Participants (%)
Any MAAE	146	52 (26.0)	60	29 (14.5)	86	35 (19.9)
MAAEs experienced by ≥2 participants, by preferred term
Acute sinusitis	10	9 (4.5)	2	2 (1.0)	8	8 (4.5)
Bronchitis	4	4 (2.0)	1	1 (0.5)	3	3 (1.7)
Dermatitis contact	4	4 (2.0)	2	2 (1.0)	2	2 (1.1)
Asthma	4	3 (1.5)	0	0	4	3 (1.7)
Contusion	4	2 (1.0)	0	0	4	2 (1.0)
Back pain	3	3 (1.5)	3	3 (1.5)	0	0
Sleep apnea syndrome	3	3 (1.5)	3	3 (1.5)	0	0
Oropharyngeal pain	2	2 (1.0)	2	2 (1.0)	0	0
Tachycardia	2	2 (1.0)	2	2 (1.0)	0	0
Dermatitis	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Hypertension	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Bacterial vaginosis	2	2 (1.0)	1	1 (0.5)	1	1 (0.6)
Cystitis	2	2 (1.0)	0	0	2	2 (1.1)
Influenza	2	2 (1.0)	0	0	2	2 (1.1)
Otitis media acute	2	2 (1.0)	0	0	2	2 (1.1)
Limb injury	2	2 (1.0)	0	0	2	2 (1.1)
Neck pain	2	2 (1.0)	0	0	2	2 (1.1)
Cervical dysplasia	2	2 (1.0)	0	0	2	2 (1.1)

Any participant who experienced >1 adverse event is counted under each vaccine dose column, but is counted only once in the “Any vaccination” column.

MAAEs were defined as adverse events leading to an unscheduled visit to, or by, a healthcare professional, including visits to an emergency department, but not fulfilling seriousness criteria. All MAAEs were evaluated for the entire duration of the trial.

Two MAAEs for one (0.5%) participant were the only ones attributed to TAK-003 and were observed after the first dose: abdominal discomfort and oropharyngeal pain. Both events were moderate in intensity, resolved, and did not result in discontinuation from the trial or withdrawal from the second TAK-003 dose.

TAK-003 withdrawal and/or participant discontinuation from the trial

None of the AEs leading to TAK-003 withdrawal and/or discontinuation from trial was attributed to TAK-003 or to trial procedures.

A total 24 participants (12.0%) prematurely discontinued the planned TAK-003 dose regimen (Figure 3b). Five (3.0%) participants who experienced 7 AEs after the first vaccine dose withdrew second TAK-003 dose administration. One event (mild back pain) failed to resolve and resulted in both withdrawal before the second dose of TAK-003 and discontinuation from the trial. Other events leading to withdrawal before second TAK-003 dose were moderate hypertension (not resolved), moderate hepatic failure (SAE, resolved), and mild events of tachycardia (not resolved), back pain (not resolved), urticaria (resolved), and abortion induced (resolved).

A total 32 (16%) participants discontinued from the trial (Figure 3b), of whom 10 withdrew consent to participate further in the trial. Eight participants withdrew consent because of non-medical reasons (e.g., relocation to another state) and the other two participants because of AEs: one withdrew consent 91 days after the first dose (no second TAK-003 dose) because of ongoing mild back pain (not TAK-003-related), and the other who withdrew consent 115 days after the first dose (no second TAK-003 dose) after diagnosis of moderate hepatic failure that was an SAE not attributable to TAK-003.

SAEs

Five (2.5%) participants experienced five SAEs. Two events were reported between the first and second vaccinations: moderate hepatic failure and severe osteoarthritis. Three other SAEs of moderate intensity (bradycardia, inguinal hernia, and sepsis) were reported in the 6-month period after the second vaccination. Sepsis was reported 2 days following the second TAK-003 dose, the event was considered not related to TAK-003 administration. All SAEs resolved and were assessed by the investigator as unrelated to vaccine administration or to trial procedures.

No deaths were reported in this trial.

Discussion

This open-label, single-arm phase 3 trial evaluated the immunogenicity and safety of naturally-aged TAK-003 in adult participants administered two doses 3 months apart during the latter half (14–20 months) of its clinical 24-month shelf-life. It was considered reasonable to omit a comparator placebo group in this trial at the time of its conception because the emerging cumulative safety data from preceding and ongoing TAK-003 trials had provided some confidence in TAK-003’s safety profile.

The emergence of SARS-CoV-2 virus and COVID-19 disease pandemic during the trial did not affect its conduct or completion, participant safety or data integrity: the last participant visit occurred 2 days after the COVID-19 pandemic was declared on 11 March 2020.²⁴

Sixty-one (31.5%) participants in this trial were excluded from the PPS immunogenicity analysis, and this is partly attributable to a sizable proportion (28 [14.1%]) of participants who were baseline dengue seropositive. This was not considered unusual as it is similar to the 15.3% adult baseline seropositive participants noted in a predecessor TAK-003 trial, DEN-304 (NCT03423173), which was also exclusively USA-based.²⁵ It is posited that the baseline seropositive participants probably acquired dengue infection inadvertently while previously traveling to dengue endemic areas. Three times as many participants at the Anaheim site, in the present trial DEN-307, compared with the Hutchinson site, were baseline seropositive to anti-DENV neutralizing antibodies. All four DENV serotypes were associated with the baseline-positive serostatus, and, although positive serostatus with DENV-1 and DENV-2 was predominant, the neutralizing antibody titers were low: <65 for DENV-1, ≤85 for DENV-2, between 12 and 38 for DENV-3, and, between 12 and 50 for DENV-4.

The results from this trial affirmed that TAK-003 elicited neutralizing antibodies against each of the four DENV serotypes in dengue seronegative adults, with the highest titers induced against DENV-2, and persistence of immune responses against all four serotypes up to 6 months after the second dose although, a decline in neutralization titers ranging from 43% to 52% was observed for all serotypes over the same period. Presently, there is no established correlate of protection for DENV unlike that of flavivirus vaccines such as yellow fever virus^26,27 and Japanese encephalitis.²⁸ Without an established correlate of protection, changes in neutralizing antibody titers do not portend a direction of effect on vaccine efficacy. Seroconversion rates for all dengue serotypes in the present study were robust and persistent (>85%) through to 6 months after the second dose. This persistency of neutralizing antibody response and maintenance of high seroconversion rates up to 6 months after the scheduled two doses, coupled with higher GMTs for the DENV-2 serotype than those for DENV-1, DENV-3, and DENV-4 is a pattern that is consistent with the data observed in the pivotal TAK-003 efficacy trial, DEN-301,^19,20,29 and other Phase 2^30–32 and 3 TAK-003 trials.^25,33–35

There was no formal hypothesis testing in this trial. Nevertheless, an informal alignment of data from this trial shows that the immune response elicited from baseline seronegative participants, as described by GMTs, is of the same order of magnitude as those observed in two TAK-003 trials (DEN-301 and DEN-304) that involved its administration without predefined aging at long-term storage conditions (Figure 6). In comparison with trial DEN-307 where TAK-003 had “aged” 14–20 months by the time the last participant was administered with the second dose, the DEN-301 trial (NCT02747927) involved >20,000 pediatric (4–16 years old) participants, for whom the vaccine material had been targeted for potency to simulate material that had been held for a period approaching the shelf-life of the product, whereas trial DEN-304 (NCT03423173) involved >900 adult (18–60 years old) participants, for whom the vaccine material had “aged” 7.5–11 months.²⁵ As evident from Figure 6 (see also Figure S1), the immune response elicited with the TAK-003 vaccine batch in trial DEN-307 against all four dengue serotypes in baseline dengue seronegative participants does not differ substantially from that elicited with the TAK-003 administered in trials DEN-301 and DEN-304. TAK-003 aging over time does not materially affect its immunogenic properties across the four serotypes and is further illustrated from the month-by-month GMTs (Figure 7) and titer (Figure S2) data for trial DEN-307 baseline seronegative participants during the course of their enrollment and dosing over the 14–20 months interval.

Figure 6. Baseline dengue seronegative participant geometric mean titers (95% confidence interval) at day 120 and day 270 after TAK-003 administration in trials DEN-301,^19,20,29 DEN-304²⁵ and DEN-307. Abbreviation: DENV, dengue virus.

Figure 7. Baseline dengue seronegative participant geometric mean titers (95% confidence interval) at day 120 and day 270 following administration of TAK-003 in trial DEN-307. Abbreviation: DENV, dengue virus.

TAK-003 was well tolerated with respect to reactogenicity (solicited local and systemic AEs), with the second dose eliciting fewer events than the first dose. The unsolicited AEs and MAAEs encountered in this trial were reflective of the age range of adult population enrolled, although the incidences of the most frequently reported unsolicited AEs here were considerably lower than those reported as background rates and frequently associated with comorbidities.^36–41 No important safety risks were identified from this trial: the safety data from this trial generally mirror that of the broader population in the TAK-003 clinical development database.⁴²

This trial demonstrated that the immune response that was elicited and persisted up to 6 months after the second dose of TAK-003 is acceptable when the product is stored as directed and when it is administered in the second half of its clinical 24-month shelf-life. The safety profile following administration of a naturally-aged TAK-003 product is consistent with that from other phase 2 and 3 trials with TAK-003, and no important safety risks were identified.

Data sharing

The datasets, including the redacted study protocol, redacted statistical analysis plan, and individual participants data supporting the results of the completed studies, will be made available within 3 months from initial request, to researchers who provide a methodologically sound proposal. The data will be provided after its de-identification, in compliance with applicable privacy laws, data protection, and requirements for consent and anonymization. Data requests should follow the process described in the Data Sharing section on https://clinicaltrials.takeda.com/and https://vivli.org/ourmember/takeda/.

Acknowledgments

This trial was funded by Takeda. Support in writing and editing this manuscript was provided by Nathaniel Holman, PhD (Excel Medical Affairs), and funded by Takeda. The authors interpreted the data independently.

The authors wish to thank the DEN-307 trial participants, trial investigators (Dr Ruhlman, Hutchinson, KS, USA, and Dr Winkle, Anaheim, CA, USA) and their respective staff for their support in conducting this TAK-003 clinical trial.

Disclosure statement

S.S.P., A.F., F.N., and C.G.-T. are employees of Takeda and own stock/stock options in Takeda. I.L. was an employee of Takeda when this trial was conducted and completed. S.S.P., A.F., F.N., and C.G.-T. report support from Takeda for attending meetings and/or travel, and stock options from Takeda.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2023.2254964.

Additional information

Funding

The work was supported by the Takeda Pharmaceuticals International.