Abstract
Background:
Morbidity and mortality from dengue virus (DENV) is rapidly growing in the large populations of South Asia. Few formal evaluations of candidate dengue vaccine candidates have occurred in India, Pakistan, or Bangladesh. Tetravalent vaccines must be tested for safety and immunogenicity in all age groups and in those previously exposed and naïve to DENV infections. TV005 is a leading live, attenuated tetravalent dengue vaccine.
Methods:
In dengue-endemic Dhaka, Bangladesh; following technology transfer, we performed a randomized, placebo-controlled age de-escalating clinical trial of TV005. Volunteers with age inclusion 1–50 years were healthy, as determined by history, clinical exam and safety labs. They were block randomized in four age groups at 3:1 to receive vaccine: placebo. Subjects were followed for three years. Per protocol analysis was performed. Primary outcomes were safety, evaluated as proportion of volunteers with solicited related adverse events of any severity through 28 days post dosing, and post-vaccination seropositivity by day 180 using serotype-specific neutralizing antibodies (PRNT50≥10). Secondary outcomes included viremia, impact of past dengue exposure and durability of antibody responses.
Findings:
192 volunteers were enrolled into four age groups (1–49 years of age). Vaccination was well tolerated; most adverse events were mild. Rash was the most common (26%) and differences in fever, arthralgia were seen. Post-vaccine, volunteers of all ages were seropositive to most serotypes; overall DENV1 (83%), DENV2 (99%), DENV3 (96%), DENV4 (87%). Viremia was not consistently found; antibody titers were higher in subjects with past dengue exposure. Although antibody titers to most serotypes remained stable in adults, titers to DENV 1,3,4 waned by three years in the youngest age cohort.
Interpretation:
With three years of follow-up, the single-dose tetravalent dengue vaccine, TV005, was well tolerated and immunogenic for all four serotypes in young children to adults, including subjects with no previous dengue exposure.
BACKGROUND
Dengue is the most rapidly spreading arboviral infection; cases and deaths are highest in South and Southeast Asia 1. The single-stranded RNA dengue virus (DENV) has four serotypes (DENV1–4), is a member of the Flavivirus family, and is transmitted by the Aedes mosquito. Initial infection with one serotype induces long-term homotypic immunity to that serotype and shorter-term heterotypic immunity to the other serotypes 2. Each DENV serotype causes a range of outcomes, including asymptomatic infection, self-limited febrile illness, and severe disease, including hemorrhagic fever/dengue shock syndrome (DHF/DSS). Most cases of severe disease occur in dengue-endemic regions and in persons experiencing a second DENV infection with a heterologous serotype 2. However, epidemiologic data demonstrates that sequential exposure to multiple serotypes confers broad protection from all serotypes. The ideal tetravalent dengue vaccine should permit long-lasting protection against clinical illness following vaccination and concurrent exposure to all four serotypes 3.
In tropical and sub-tropical regions, dengue poses a major public health threat, particularly in urban and semi-urban areas4. In 2013, dengue was estimated to cause 390 million DENV infections annually, with 100 million cases of symptomatic disease of any severity 1. Cases reported to the WHO have increased 8-fold in the past two decades4. This includes the Asia-Pacific region, with more than half of the global population and over 70% of the burden of dengue. Annual outbreaks are increasing in size, frequency, and severity in the large populations in Pakistan, India, and Bangladesh 1,5–7. In 2018, India reported 101,192 cases including several dengue epidemics representing all DENV serotypes 5. In Bangladesh, since the first reported dengue outbreak (DENV3) in 1964 8, outbreaks have become serious annual events with thousands of hospitalizations, including children6. In addition to DENV3, which has been implicated in multiple large outbreaks8–10, DENV1 and DENV2 have circulated11,12, but until the introduction of new diagnostic capabilities, the specific circulating serotypes of DENV were often unknown year to year.
Development of dengue vaccines targeting all four serotypes is a top priority of the World Health Organization. Because of the risk of severe disease with secondary infections, the ideal dengue vaccine should elicit a robust homotypic, and long-lived immune response to all serotypes of DENV 3. The first dengue vaccine to reach large-scale testing and receive licensure in several countries was Dengvaxia® (Sanofi Pasteur), a chimeric live-attenuated tetravalent dengue vaccine consisting of structural genes (M, membrane and E, envelope) from DENV1–4 on a genetic construct consisting of yellow fever virus YF-17D vaccine non-structural genes. This 3-dose vaccine demonstrated an overall efficacy of 30.2%−59.2%, with a reduced efficacy against DENV-2 at one year following the third dose13. Children who were DENV sero-naive at the time of vaccination were found to have an elevated risk of hospitalization upon subsequent dengue exposure; likely due to a lack of infection/immunity across all serotypes 14,15. Use in several countries is now restricted to individuals with a previous history of DENV infection or in areas with high (>80%) seroprevalence 16. A second vaccine that has recently received licensure in some countries is the two-dose Takeda tetravalent dengue (TAK-003 or QDENGA®) vaccine using a DENV-2 background. In phase III trials, it demonstrated (in 4–16-year-old children) an overall efficacy of 76.1% in DENV previously exposed participants (seropositive) over 17 months post-vaccine, and 66.2 % in those seronegative at baseline 17. However, vaccine efficacy in naïve children ages 4–5 was only 22.9% in phase III trials 18 and there were an excess number of hospitalized DENV-3 cases in the vaccinated group in year 3 19. Similar concerns have been raised due to the immunodominance of the DENV-2 component of this vaccine20.
The vaccine studied herein, TV005, was developed by the U.S. National Institute of Allergy and Infectious Diseases (NIAID, NIH), as a single dose, tetravalent, live attenuated dengue vaccine candidate. Both TV005 and a closely related vaccine (TV003) have undergone extensive Phase I and II trials in dengue-naïve adults in the United States and results from multiple clinical trials and controlled human infection models have been reported 21. In a challenge study, TV003-vaccinated volunteers subsequently exposed to recombinant DENV2 challenge strains had no evidence of infection (as measured by viremia) 22. Robust field efficacy data was recently released from Brazil and indicated an overall efficacy of 80% for two years following vaccination with TV003 23. Both the TV003 and TV005 admixtures are comprised of four serotype-specific dengue vaccine candidates: rDEN1Δ30, rDEN2/4Δ30(ME), rDEN3Δ30/31, and rDEN4Δ30; TV005 contains a 10-fold higher dose of the DENV-2 component compared to TV003 21. Safety data of TV005 demonstrated that mild rash is the only adverse event with a higher incidence in vaccinees versus placebo recipients 24. Asymptomatic low-level vaccine-associated viremia is demonstrated in most vaccinated US volunteers, when frequent testing post-vaccination is performed, and broad neutralizing antibodies are induced to all four serotypes 21,25.
Despite the enormous populations (1.8 billion persons) at-risk for dengue in the Indian subcontinent, clinical trials of tetravalent dengue vaccines had not been performed or published from India, Bangladesh or Pakistan prior to the start of this trial in 2016. Only Sri Lanka (population 22 million) had hosted dengue vaccine trials (as well as several sites in Southeast Asia) 17. To establish the capacity building to perform formal dengue vaccine trials and assess serotype-specific dengue surveillance, we began the Dengue-in-Dhaka Initiative (DIDI) in 2015. This included extensive technology transfer and validation of core dengue assays, vaccine preparation, as well as dengue vaccine clinical trial and regulatory expertise. Following a cross-sectional seroprevalence study to estimate dengue exposure in all ages, the Phase II clinical trial reported herein was performed to determine the safety, immunogenicity, and 3-year durability of the TV005 tetravalent live attenuated dengue vaccine. Since the safety of a dengue vaccine is critical in all ages, the study was a randomized, age de-escalation, double-blind, placebo-controlled study with four age cohorts. Our results suggest that regardless of age and evidence of previous baseline DENV exposure, TV005 is well-tolerated and prompts a robust multi-serotype immune response, establishing TV005 as a promising tetravalent dengue vaccine for South Asian and all global populations.
METHODS
Study Oversight
Research was approved by the institutional review boards at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and the University of Vermont. Informed consent was obtained in accordance with federal and international regulations (21CFR50, ICHE6). The vaccine trial was performed under an investigational new drug application (#14268) reviewed by the Food and Drug Administration and approved by Institutional review boards at the icddr,b and UVM. Prior to age de-escalation, all safety data thru study day 28 were reviewed by external independent Data Safety Monitoring Boards from the trial sponsor (NIAID) and the icddr,b. Clinicaltrials.gov: NCT02678455
Trial Designs and Study Setting
A Phase II randomized, placebo-controlled vaccine trial was conducted in Mirpur, a densely populated region of Dhaka, Bangladesh, to examine the safety and immunogenicity of TV005. An icddr,b-led household census of the study area was performed to identify eligible individuals and to explain the study to the community. Volunteers were enrolled between March 2016 and February 2017 and followed for three years post-injection. Primary study outcomes were vaccine safety and immunogenicity. Safety was measured by solicited vaccine-related adverse events (AEs) of any severity, onset by day 28 post-vaccine. Immunogenicity outcome was % seropositivity among vaccinated volunteers (overall, by age and serotype), through day 180 post-vaccination. Neutralizing antibody levels to DENV1–4 were measured at days 0, 28, 56, and 180, 360, 720 and 1080 post-vaccination to measure immunogenicity (up to day 180) and durability of response (a secondary outcome, day 360–1080). Day 14 was drawn for adults/adolescents only. Other secondary objectives were vaccine viremia (occurrence, maximum titer, duration) and description of the impact of previous dengue exposure on seropositivity post-vaccine. Exploratory outcomes evaluated the geometric mean titers of neutralizing antibody responses, as well as seropositive to number of serotypes.
Sample Size: With 36 vaccinated participants per age cohort, the study has 80% power to detect an adverse event that occurs in around 1 in 20 participants. Age de-escalation design was necessary per the Data Safety Monitoring Board (DSMB), and sample size was selected building upon previous Phase I and II studies of each monovalent candidate and tetravalent admixtures, in the United States and Thailand, which used similar cohorts and similar vaccine to placebo ratios.
Randomisation and Masking: Volunteers were randomized stratified by cohort 3:1 vaccine: placebo in balanced blocks of size 4. The study was double blind and was unmasked at day 180; outcome assessors, clinic staff, and volunteers remained blind throughout.
Study Population
All study participants were recruited from Wards, 2, 3, and 5 of the Mirpur area of Dhaka, Bangladesh. The trial enrolled healthy volunteers (inclusion criteria ages 1–50) years into four age cohorts (years); adults (18–50), adolescents (11–17), children (5–10), and young children (1–4). Eligibility criteria included, though was not limited to, lack of chronic diseases, seronegativity to Hepatitis B and C, normal hematology and serum chemistry, and a normal physical exam. Women of reproductive age could be neither breastfeeding nor pregnant and were required to use approved contraceptive methods for 28 days following vaccination. Subjects were enrolled agnostic to past exposure to DENV.
Vaccines
Previous clinical trials of TV005 describe the safety, immunogenicity, attributes of viral replication, as well as safety data in non-human primates25. Prior to use, the single serotype vaccine viruses were stored at −80°C ± 15°C and then thawed, diluted, and combined as an admixture immediately prior to vaccination. Vaccines were diluted to 3.3 log10/mL plaque-forming units (PFU) for serotypes DENV-1, 3 and 4 while the DENV-2 vaccine was diluted to 4.3 log10/mL PFU. Viral titers were confirmed following the preparation of all admixtures. Placebo inoculations consisted of diluent only (Leibowitz’s L-15 medium, Millipore Sigma, Burlington, MA).
Clinical Trial Procedures and Evaluation
On study day 0, volunteers were randomized to receive vaccine or placebo in a 3:1 ratio. Vaccine was administered by subcutaneous injection of a 0.5-mL dose. Volunteers had daily inhome visits through study day 14 (and weekly visits to day 28) with measurement of oral temperature and assessment of AEs. At the clinic, clinical assessments, blood for serology, and physical examinations were performed on study days 7, 14, 28, 56, 180, 360, 720, and 1080, and as needed. Viremia assessment was done 7- and 14-days post-vaccination (day 14 was not collected for the youngest two age cohorts). AEs were captured and recorded through study day 28 with all followed to resolution. Febrile surveillance and monitoring for SAE continued for three years with temperature monitoring by volunteers, weekly home visits by staff, and evaluation for dengue (NS1 and IgG/IgM testing) and other febrile illnesses. AEs were graded for severity and relationship to vaccination using protocol-defined grading and standard toxicity tables. Dengue vaccine-like rash was defined as a maculopapular rash, typically on the trunk and proximal extremities. 24
Serologic Response and Virus/Viremia Quantitation
Serologic responses (our measure of immunogenicity) and virus quantitation were determined as previously described 24. Serum was tested at times noted above except day 14 was not collected for the youngest two age cohorts. For both studies neutralizing antibody response was measured with standard 50% plaque-reduction neutralization test (PRNT50) where the lowest titer showing 50% plaque reduction was calculated, using an initial dilution of 1:5 and 4-fold serial dilutions. A seropositive response was defined as a PRNT50 value ≥10. Viremia was determined by amplification and direct titration using Vero cell monolayers, as previously described 24.
Data Analysis
Per-protocol analysis was performed. Proportion of solicited treatment related adverse events in TV005 versus placebo recipients were estimated by proportion difference and 95% confidence intervals, adjusted by age cohort. Fisher’s exact test was used to determine statistically significant differences in percentage of seropositive to each serotype between dengue-experienced and dengue-naïve study participants. Statistical analysis was performed using SPSS (version 28.0), SAS 9.4, and R Core Team software (version 4.2.2). Neutralizing antibody titers are presented as reciprocal geometric mean titers.
The role of the study funder: Study funders, NIH and Johns Hopkins University, had no role in study design, or in collection, analysis and interpretation of data. They had no role in writing the manuscript or the decision to submit for publication.
RESULTS
The TV005 Phase II live attenuated dengue vaccine trial assessed 648 potential volunteers for eligibility. Major reasons for exclusion varied between age cohorts and included baseline anemia, other underlying acute or chronic medical conditions, and malnutrition. As shown in Figure 1, 192 individuals were enrolled and randomized in four age groups of 48 persons (range: 1–49 years). Using within-cohort 3:1 vaccine: placebo randomization, 144 volunteers received a single dose of TV005 and 48 received placebo. Protocol deviations were infrequent and included <1% of all scheduled visits and specimens missed or out of window. Demographics and ages of the study population are shown in Table 1.
Figure 1 – CONSORT Flow diagram of eligibility, enrollment and receipt of TV005 or placebo.
Table 1:
Demographics of 192 enrolled participants, TV005 phase II trial.
| TV005 n=36 | Placebo n=12 | ALL n=48 | |
|---|---|---|---|
| Age (years) | |||
| Adults (18–50 years) | 34.7 ± 16.4 | 29.2 ± 8.1 | 33.4 ± 8.2 |
| Adolescents (11–17 years) | 12.5 ± 1.8 | 12.8 ± 1.6 | 12.6 ± 1.8 |
| Children (5–10 years) | 8.1 ± 1.4 | 7.8 ± 1.3 | 8.1 ± 1.4 |
| Young children (1–4 years) | 3.3 ± 0.8 | 3.4 ± 0.5 | 3.3 ± 0.7 |
| All | 14.7 ± 14.5 | 13.3 ± 10.7 | 14.3 ± 13.5 |
| Sex (Male) | |||
| Adults (18–50 years) | 13 (36%) | 7 (58%) | 20 (42%) |
| Adolescents (11–17 years) | 19 (53%) | 8 (66%) | 27 (56%) |
| Children (5–10 years) | 10 (28%) | 5 (42%) | 15 (31%) |
| Young children (1–4 years) | 22 (61%) | 7 (58%) | 29 (60%) |
| All | 64 (44%) | 27 (56%) | 91 (47%) |
Data are mean with SD and n(%). All subjects were Bangladeshi (100%).
TV005 was well-tolerated. Local and systemic solicited AEs are shown in Table 2, by age cohort. With the exception of rash, few adverse events were noted overall, most were generally mild and of short duration. An asymptomatic dengue vaccine-like rash (neither pruritic nor painful) occurred more frequently in vaccinees than placebo recipients (estimated adjusted proportion difference 17%, 95 % CI 2–28%). A total of 37 episodes of rash were observed; 36 were asymptomatic (grade 1 events); with one pruritic rash (grade 2). The median onset of rash was day 9 (interquartile range, 7–14 days) after vaccination with a median rash duration of 6 days (interquartile range, 3–14 days). There were four episodes of thrombocytopenia assessed as related to TV005. Three events were mild (grade 1). One was an asymptomatic grade 3 event (40,000 m3/mL) in a 4-year-old child. However, platelets rebounded to normal (240,000 m3/mL) within three days. Following consultation with a Pediatric Hematologist, this event was determined to be due to platelet clumping in specimen management. Seven cases of fever were noted between day 0–17, six were mild (38–38.5°C) and ≤2 days in duration; one was moderate (38.6°C) and lasted one day. Arthralgia was seen in seven vaccinees: four were mild (<1 day to 1 day) and three were moderate (2–6 days).
Table 2:
Solicited Adverse Events related to TV005 vaccination, overall and by age cohort.
| Total | Adults | Adolescents | Children | Young Children | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pooled TV005 n=144 | Pooled Placebo n=48 | Adjusted Proportion Difference (95% CI) | TV005 n=36 | Placebo n=12 | Proportion Difference (95% CI) | TV005 n=36 | Placebo n=12 | Proportion Difference (95% CI) | TV005 n=36 | Placebo n=12 | Proportion Difference (95% CI) | TV005 n=36 | Placebo n=12 | Proportion Difference (95% CI) | |
| Local Injection Site | |||||||||||||||
| Erythema | 1 (1%) | 0 (0%) | 0.028 (−0.216 – 0.141) | – | – | – | – | – | – | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | – | – | – |
| Swelling | 1 (1%) | 0 (0%) | 0.028 (−0.216 – 0.141) | – | – | – | – | – | – | – | – | – | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) |
| Laboratory | |||||||||||||||
| ALT Increased | 5 (3%) | 2 (4%) | 0.028 (−0.135 – 0.119) | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) | 1 (3%) | 1 (8%) | −0.055 (−0.327 – 0.077) | 2 (6%) | 1 (8%) | −0.027 (−0.301 – 0.115) | – | – | – |
| Hemoglobin Decreased | 5 (3%) | 2 (4%) | 0.028 (−0.135 – 0.119) | – | – | – | 1 (3%) | 1 (8%) | −0.055 (−0.327 – 0.077) | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) | 2 (6%) | 1 (8%) | −0.027 (−0.301 – 0.116) |
| Leukocytosis | 2 (1%) | 1 (2%) | 0.020 (−0.205 – 0.121) | – | – | – | 1 (3%) | 1 (8%) | −0.055 (−0.327 – 0.077) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | – | – | – |
| PTT Prolonged | 4 (3%) | 2 (4%) | 0.024 (−0.127 – 0.111) | – | – | – | 3 (8%) | 0 (0%) | 0.083 (−0.165 – 0.218) | 0 (0%) | 1 (8%) | −0.083 (−0.354 – 0.035) | 1 (3%) | 1 (8%) | −0.055 (−0.327 – 0.077) |
| Thrombocytopenia | 4 (3%) | 0 (0%) | 0.056 (0.022 – 0.134) | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) | – | – | – | – | – | – | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) |
| Systemic | |||||||||||||||
| Fever | 7 (5%) | 0 (0%) | 0.051 (0.018 – 0.118) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 3 (8%) | 0 (0%) | 0.083 (−0.165 – 0.218) | – | – | – | 3 (8%) | 0 (0%) | 0.083 (−0.165 – 0.218) |
| Headache | 17 (12%) | 7 (15%) | −0.036 (−0.203 – 0.089) | 4 (11%) | 2 (17%) | −0.055 (−0.345 – 0.130) | 7 (19%) | 3 (25%) | −0.056 (−0.354 – 0.169) | 6 (17%) | 2 (17%) | 0 (−0.295 – 0.194) | – | – | – |
| Rash | 37 (26%) | 6 (12%) | 0.166 (0.016 – 0.275) | 12 (33%) | 2 (17%) | 0.167 (−0.144 – 0.369) | 10 (28%) | 3 (25%) | 0.028 (−0.279 – 0.256) | 7 (19%) | 1 (8%) | 0.111 (−0.176 – 0.281) | 8 (22%) | 0 (0%) | 0.222 (−0.042 – 0.381) |
| Systemic (Not young children) | n=108 | n=36 | |||||||||||||
| Arthralgia | 7 (6%) | 0 (0%) | 0.063 (0.030 – 0.126) | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) | 3 (8%) | 0 (0%) | 0.083 (−0.165 – 0.218) | 2 (6%) | 0 (0%) | 0.056 (−0.190 – 0.181) | |||
| Fatigue | 7 (6%) | 2 (6%) | 0.023 (−0.155 – 0.118) | 2 (6%) | 1 (8%) | −0.027 (−0.301 – 0.115) | 4 (11%) | 1 (8%) | 0.028 (−0.151 – 0.185) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | |||
| Myalgia | 7 (6%) | 1 (3%) | 0.041 (−0.105 – 0.105) | 1 (5%) | 0 (0%) | 0.028 (−0.216 – 0.142) | 2 (6%) | 1 (8%) | −0.028 (−0.310 – 0.116) | 4 (11%) | 0 (0%) | 0.111 (−0.140 – 0.253) | |||
| Nausea | 6 (6%) | 2 (6%) | 0.019 (−0.160 – 0.114) | 5 (14%) | 1 (8%) | 0.056 (−0.226 – 0.218) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 0 (0%) | 1 (8%) | −0.083 (−0.354 – 0.035) | |||
| Photophobia | 1 (1%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | – | – | – | – | – | – | |||
| Retro-orbital Pain | 3 (3%) | 1 (3%) | 0.024 (−0.142 – 0.088) | 1 (3%) | 1 (8%) | −0.055 (−0.327 – 0.077) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | |||
| Systemic (Young children only) | n=36 | n=12 | |||||||||||||
| Decrease in Activity | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | |||||||||
| Loss of Appetite | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | 1 (3%) | 0 (0%) | 0.028 (−0.216 – 0.141) | |||||||||
| Vomiting | 3 (8%) | 0 (0%) | 0.083 (−0.165 – 0.218) | 3 (8.3) | 0 (0%) | 0.083 (−0.165 – 0.218) | |||||||||
Note: Adjusted proportion difference (TV005 minus Placebo) with inverse variance weighting and Newcombe Score confidence intervals. Cohorts without events do not contribute to the effect estimate. Solicited adverse events do not appear in the table in the case of no recorded events in any subject. These include injection site pain, injection site pruritus, neutropenia, PT prolonged (all ages); fussiness, and drowsiness (young child cohort only).
An episode of elevated alanine transaminase (peak of 198 U/L day 14 post-vaccination) observed in an 8-year-old child and was determined to be ‘possibly related’ to vaccination. This child had a respiratory tract infection and received antibiotics and paracetamol four days after vaccination. They remained asymptomatic and ALT normalized by day 21. Four serious adverse events (SAE) unrelated to vaccine occurred, one episode each of appendicitis, angina, endocarditis, and typhoid fever.
For immunogenicity, before vaccination, baseline serologic assays (PRNT50) demonstrated that 46% (87 of 190) evaluable subjects had previous exposure to any DENV serotype, highly related to age: adults (74%), adolescents (45%), children (44%) and young children (21% [data not shown]. Following TV005 vaccination, as shown in Table 3, 118/142 (83%), 141/142 (99%), 137/142 (96%) and 124/142 (87%) of all individuals were seropositive to DENV-1, 2, 3, and 4, respectively. All ages had robust responses to all serotypes, including the youngest children who were seropositive to DENV1 (30/36; 83%;), DENV2 (36/36; 100%), DENV3 (33/36; 92%) and DENV (34/36; 94%). When evaluated by previous dengue experience (Table 4), all (100%, 61/61) volunteers with past exposure were seropositive to DENV 1,2,3 and 87% to DENV4. In dengue-naïve vaccine recipients (all ages), seroconversion was 70%, 99%, 94%, 88% for DENV 1–4, respectively Notably, although the numbers of volunteers in each group are small, 24/30 (80%) of the youngest children seroconverted to DENV1, greater than 4/9 (44%) of previously-naïve adults (Table 4, footnote). Neutralizing antibody titers for the volunteers in Table 3 and 4 are shown in Appendix Table 1 and 2. Notably, titers were substantially higher (10–15-fold) for DENV-1–3 in the experienced compared to the naïve vaccines, with 1.6-fold higher for DENV-4. As shown in Appendix Table 3, overall, 96% of volunteers (136/142) had a trivalent or better response and 71% (101/142) had a tetravalent response following vaccination. All dengue-experienced vaccinees had a trivalent or better response and 87% (53/61) had a tetravalent response to vaccination. Among naïve vaccinees, 93% (75/81) had a trivalent or better response, and 59% (48/81) had a tetravalent response.
Table 3:
Neutralizing antibody seropositivity status of TV005 vaccine recipients, at baseline and following TV005 vaccination until day 180, by age cohort and DENV serotype.
| Frequency and percent of volunteers seropositive at baseline | Frequency and percent of volunteers seropositive following TV005 | |||||||
|---|---|---|---|---|---|---|---|---|
| Age Group | DENV-1 | DENV-2 | DENV-3 | DENV-4 | DENV-1 | DENV-2 | DENV-3 | DENV-4 |
| Adults | 23/35 (66%; 49–79) | 24/35 (69%; 52–81) | 25/35 (71%; 55–84) | 7/35 (20%; 10–36) | 30/35 (86%; 71–94) | 34/35 (97%; 85–99) | 34/35 (97%; 85–99) | 32/35 (91%; 78–97) |
| Adolescents | 9/35 (26%; 14–42) | 12/35 (34%; 21–51) | 13/35 (37%; 23–54) | 0/35 (0%; 0–10) | 29/35 (83%; 67–92) | 35/35 (100%; 90–100) | 34/35 (97%; 85–99) | 30/35 (86%; 71–94) |
| Children | 11/36 (31%; 18–47) | 12/36 (33%; 20–50) | 13/36 (36%; 22–52) | 4/36 (11%; 4–25) | 29/36 (81%; 65–90) | 36/36 (100%; 90–100) | 36/36 (100%; 90–100) | 28/36 (78%; 62–88) |
| Young Children | 4/36 (11%; 4–25) | 4/36 (11%; 4–25) | 3/36 (8%; 3–22) | 1/36 (3%; 0–14) | 30/36 (83%; 68–92) | 36/36 (100%; 90–100) | 33/36 (92%; 78–97) | 34/36 (94%; 82–98) |
| ALL | 47/142 (33%; 26–41) | 52/142 (37%; 29–45) | 54/142 (38%; 30–46) | 12/142 (8%; 5–15) | 118/142 (83%; 76–88) | 141/142 (99%; 96–100) | 137/142 (96%; 92–98) | 124/142 (87%; 81–92) |
Data are n/N (%; 95% CI).
Table 4:
Frequency of neutralizing antibody seropositivity of TV005 vaccine recipients, at baseline and following TV005 vaccination, based on past exposure to DENV, and by DENV serotype.
| Baseline | Post TV005 Vaccination | |||||||
|---|---|---|---|---|---|---|---|---|
| Serotype | DENV-1 | DENV-2 | DENV-3 | DENV-4 | DENV-1 | DENV-2 | DENV-3 | DENV-4 |
| Experienced | 47/61 (77%; 6586) | 52/61 (85%; 74–92) | 54/61 (88%; 7894) | 12/61 (20%; 12–31) | 61/61 (100%; 94–100) | 61/61 (100%; 94–100) | 61/61 (100%; 94–100) | 53/61 (87%; 76–93) |
| Naïve | 0/81 (0%; 0–4) | 0/81 (0%; 0–4) | 0/81 (0%; 0–4) | 0 /81 (0%; 0.0–4) | 57/81 (70%; 60–79) | 80/81 (99%; 93–100) | 76/81 (94%; 86–97) | 71/81 (88%; 79–93) |
Note: Data are n/N (%, 95% CI). Seropositive response was defined as a PRNT50 value ≥10 at any time point (Day 14, 28, 56 or 180). Logistic model to adjust for age cohort provides unstable estimates due to quasi separation of data. Comparison between experienced vs. dengue naïve volunteers post-TV005: DENV1 (P<0.001), DENV2 (P=1.00), DENV3 (P=0.070), DENV4 (P=1.000) Note that DEN1 difference in seropositivity is primarily due to adults vs. young children. DENV-1 naïve adults who became seropositive were 4/9 (44%), adolescents 15/21 (71%), children 14/21 (67%) and young children 24/30 (80%).
Due to cultural constraints, vaccine-viremia was only measured at 7- and 14-days post-vaccination in adult/adolescent cohorts and at day 7 for child and young child cohorts; this was insufficient to detect all episodes of viremia. Viremia was not detected in any placebo recipient. By viral amplification in Vero cells, viremia was detected in six (4.2%) vaccinees, all at the lowest level of detection (maximum virus titer 0.5 log10 PFU/mL) for all (data not shown).
Post-vaccination, participants were followed by active, febrile surveillance for three years to determine long-term safety and durability of the neutralizing antibody response. During this period, additional adverse events related to vaccination were not observed. Breakthrough DENV infections were not observed among vaccine recipients, two cases (neither requiring hospitalization) were identified in placebo recipients. As shown in Figure 2, through the 3-year follow-up period, most vaccinated adults and adolescents remained seropositive to all serotypes, especially DENV2 and 3. For the progressively younger age group, many of whom were sero-naïve at baseline, seropositivity waned for most serotypes (with the exception of DENV2). By year 3, less than half of older (age 5–11yr) children were seropositive for DENV1 and 4. For younger children, approximately 25% remained seropositive for DEN 1,3,4. Given cohort sizes, this data could not be further analyzed by previous dengue exposure.
Figure 2. Post TV005-vaccination: seropositivity over time in all age cohorts, and to all dengue virus serotypes.
DISCUSSION
During 2016–2020, we performed the first clinical trial of a live-attenuated tetravalent dengue vaccine in urban Bangladesh, a region increasingly and significantly impacted by the morbidity and mortality of dengue 1,5,6. Using NIAID’s live attenuated, single-dose tetravalent dengue vaccine TV005, we demonstrate this vaccine’s safety and immunogenicity in an endemic setting. We evaluated multiple age groups (1–49 years), persons with and without previous dengue-exposure, and performed three years of follow-up. Importantly, this work, performed using standardized assays and harmonized clinical and laboratory protocols developed during the past twenty-five years of research on this vaccine (and all performed at the icdddr,b for this research) helps to build the local infrastructure for highly needed large, phase I-III trials of tetravalent dengue vaccines in Bangladesh and South Asia. Since the start of this work, additional early phase 1 trials of this vaccine have been performed in India and larger scale trials are planned26. This work reiterates specific issues of importance for dengue vaccines in endemic settings, especially age and previous DENV exposure.
Prior to the execution of the phase II trial, our Dengue-in-Dhaka-Initiative (DIDI) transferred dengue vaccine trial expertise to icddr,b partners; including training in the conduct of clinical dengue vaccine trials, regulatory expertise, and all standardized core assays and vaccine preparation. This included laboratory capacity building to obtain serotype-specific serologic data for Dhaka. To gather insights into age of exposure and circulating serotypes, and to inform the design of the subsequent TV005 clinical trial, we first performed a cross-sectional seroprevalence study. This work demonstrated that most adults (68%) had past exposure, especially to DEN 2,3 with seroprevalence diminishing by age to <10% in children < 4 years of age. 27
This TV005 Phase 2 trial presented here demonstrates the tolerability and immunogenicity of the single-dose NIAID tetravalent TV005 dengue vaccine across ages from young children to adults, enrolled agnostic to their previous exposure to DENV. As in past evaluations of this vaccine, many volunteers have very few symptoms; the most common vaccine-associated adverse event is a self-limited, rash, often not noticed by the individual. A few volunteers across age cohorts had low-grade fever, thrombocytopenia, and arthralgia; most were mild and short-lived. In a 4-year-old, safety monitoring identified thrombocytopenia but with extremely rapid (3 day) rebound which, upon consultation with a Pediatric Hematologist, was deemed due to platelet clumping during blood processing. Another child (age 8), who had received antibiotics for an upper respiratory infection and demonstrated elevated alanine transaminase (ALT), which self-resolved. For both observations, causal relation to vaccine is unlikely, but will be monitored in future studies with children.
Unlike previous studies, the direct determination of vaccine infectivity by the measurement of vaccine viremia was not possible in this study due to limitations placed on blood collection frequency and volumes during the periods of expected viremia. Nevertheless, most subjects mounted a neutralizing antibody to all four DENV serotypes following vaccination at levels greater than that associated with seropositivity (PRNT50≥10). 83.1% of individuals overall were seropositive to DENV-1, the lowest response, and 99.3%, 96.5% and 87.3% were seropositive to DENV-2, 3, and 4, respectively. A trivalent response was demonstrated in 96%, and a tetravalent response was seen in 71% of subjects overall. Past research on TV003 and TV005 has demonstrated that each of the four serotype within the tetravalent vaccine formulation is infectious, prompting the production of homotypic (serotype-specific) antibodies22,25,28. Thus, the broad tetravalent response is hypothesized to indicate the capacity of each serotype to infect and drive these specific responses.
Past exposure to DENV (“dengue-experienced”) was defined herein as pre-existing neutralizing antibodies (PRNT50≥10) before receipt of vaccine or placebo. As in many endemic settings, dengue experience is highly correlated with age. Post-TV005 vaccination neutralizing antibody titers (except for DENV-4) were significantly higher in dengue-experienced subjects (up to 15-fold) than in dengue-naïve subjects. Based on pre-vaccination low antibody titers (in all ages) to DENV-4 (some of which may be cross-reactive from other serotypes in older age groups), we suspect this serotype had not yet circulated in this region. In agreement, DENV-4 was not detected by molecular testing in Bangladesh (including Dhaka) in outbreaks spanning 2013-2019, partially encompassing the timeframe in which this trial took place10–12.
Dengue-naïve persons are at the highest risk for wild-type or vaccine-induced severe dengue disease due to heterotypic infection and antibody dependent enhancement 3,16. This may also occur if a dengue vaccine is biased toward a single serotype, similar to a monovalent infection. Our data demonstrates that after a single dose of TV005, the majority (87%) of dengue-experienced subjects demonstrated robust responses to all four serotypes suggesting that for this population (as in our other studies), TV005 prompts homotypic antibody responses to the remaining serotypes which have never infected the individual, increases antibodies to all serotypes previously seen, and promotes durable protection from all serotypes.
We demonstrate that circulating antibody titers are durable for three years in adults and adolescents. The decay of circulating neutralizing antibodies in young children (most of whom have never been exposed to Dengue) is noted, and not surprising. At three years, only approximately 25% of the youngest children were seropositive to DENV 1,3,4, from their peak post-vaccination (83% DENV1, 100% DENV2, 92% DENV3, 94% DENV4). Circulating antibodies cannot reflect the impact of other immune components, especially memory B cells in protection from disease. As below, the role of memory B cells, as well as other components of immune protection, are still under study in this population. Multiple studies of DENV infection and the NIAID tetravalent dengue vaccine demonstrate the importance of the dengue-specific multifunctional cytokine-producing T cells targeting non-structural proteins, as well as memory B cell responses 29,30. The significant of other these immune components will be necessary to determine whether a second dose of TV005 is necessary for the durable protection of young (seronaive) children.
Overall, the results of the phase II evaluation of the NIAID tetravalent TV005 dengue vaccine are promising for its large-scale use in outbreak and endemic settings, for both dengue-experienced and dengue-naïve individuals, including children. This vaccine, as well as TV003 (the same components but with one-log lower DENV2), are now licensed for further development to multiple pharmaceutical companies, including Merck, The Serum Institute of India, Butantan, Panacea, and others (none of whom had a role in this study). As compared to other licensed or candidate tetravalent vaccines, a single dose of the NIAID tetravalent TV005 vaccines prompts an immune response across all serotypes post-vaccination.
This work has multiple limitation, including the small sample size in each category of age and past dengue experience. These small sizes prohibited detailed current analyses of ages and exposure categories. Due the inability to draw blood frequently, as was done in early studies in the United States, viremia due to vaccination is likely underestimated and viremia data cannot be used with confidence. Our febrile surveillance may have also missed acute dengue cases in our three years of follow-up, although we are certain that no dengue hospitalizations or deaths occurred. As noted, other non-neutralizing antibody components of protective immunity, such as memory B-cell populations, CD4/CD8 non-structural protein and innate responses, may contribute to protective immunity, and are currently under investigation.
Ongoing additional research to evaluate clinical protection includes a dengue vaccine-human challenge model study in Bangladeshi adults, and continued follow-up of the large phase III efficacy trial in Brazil. Although additional supporting data is needed, especially in the youngest persons, this work represents the start of our DIDI initiative to accelerate dengue vaccine trials and dengue vaccine access for the large populations of the Indian subcontinent, as well as other endemic regions with high burdens of illness caused by dengue viruses.
Supplementary Material
Acknowledgements
The Dengue in Dhaka Initiative (DIDI) team would like to thank the Dhaka City Corporation for their continued support, the volunteers and families for their participation, as well as the efforts of the full study team.
Funding Sources:
The funders of the study were the National Institutes of Health and University of Vermont. They had no role in study design; collection, analysis and interpretation of data; writing of the report, and the decision to submit for publication.
Footnotes
Funding: NIH NIAID Intramural Research Program contracts # HHSN272200900010C, 75N93019D00031; Johns Hopkins University.
Authors who are full professors or the equivalent are: KK Pierce, F Qadri, AP Durbin, SS Whitehead, R Haque and BD Kirkpatrick
Results presented in part at the American Society of Tropical Medicine (ASTMH) annual meetings: Philadelphia, PA, November 2015 (abstract 1411); New Orleans, LA, November 2018 (abstract 2830) and National Harbor, MD, November 2019 (abstract 1479).
Declaration of Interests: Walsh MCR, Pierce KK, Carmolli M, Dickson DM, Bak DM, Diehl SA, Nazib F, Kirkpatrick BD are employed by the University of Vermont which was the recipient of research funding from the NIH and Johns Hopkins for this work. Whitehead SS is employed by the National Institutes of Health, which partially funded this work. Durbin AP is employed by Johns Hopkins University, which receives funding from the NIH for dengue research. Alam MS, Alam M, Afreen S, Golam K, Qadri F, Haque R are employed by the icddr, b which received funding from UVM for the conduct of this research.
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Data Sharing:
Requests for access to relevant data underlying the results of this study will be made available 6 months after publication upon request. Submission of a proposal will be granted to access to de-identified individual participant data after review and approval by sponsor, investigator, and collaborators based on scientific merit. Proposals should be sent to the corresponding author. A data access agreement will be required, and data will be shared through a secure online platform.
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Associated Data
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Supplementary Materials
Data Availability Statement
Requests for access to relevant data underlying the results of this study will be made available 6 months after publication upon request. Submission of a proposal will be granted to access to de-identified individual participant data after review and approval by sponsor, investigator, and collaborators based on scientific merit. Proposals should be sent to the corresponding author. A data access agreement will be required, and data will be shared through a secure online platform.