Safety and Immunogenicity of Recombinant, Live Attenuated Tetravalent Dengue Vaccine (CYD- TDV) in Healthy Vietnamese Adults and Children

Background: Dengue viruses (DENV1-4) are estimated to infect 50-100 million individuals per year worldwide including an estimated 500,000 people with severe dengue who require hospitalization every year. The live, attenuated, tetravalent dengue vaccine (CYD-TDV) candidate, containing four recombinant dengue viruses (CYD14), is in clinical phase III. Methods: In an observer-blind, phase II trial in Long Xuyen, Vietnam, 180 children and adults (range: 2-45 years) were randomized 2:1 to receive 3 CYD-TDV vaccinations at months (M) 0, 6 and 12 or meningococcal polysaccharide A+C at M0, placebo at M6, and typhoid Vi polysaccharide at M12. Serum antibody responses against the CYD1-4 parental wild-type dengue viruses were assessed using plaque-reduction neutralization test (PRNT50). Safety and reactogenicity were assessed using conventional methods. Febrile episodes lasting ≥ 48 h with suspicion of dengue (passive surveillance) were virologically confirmed. (ClinicalTrials.gov: NCT00875524). Results: At baseline 139(77%) were seropositive (titer ≥ 10 l/dil) against dengue or Japanese encephalitis; 36% were seropositive to all four dengue serotypes. After the first CYD-TDV vaccination, 53% were seropositive to all four serotypes, increasing to 72% and 92% after the second and third vaccinations. In the control group seropositivity against all four dengue serotypes was 28% at baseline and slightly increased at 36% after the third injection 13 months later. After the third CYD-TDV vaccination, 96% were seropositive to at least 3 serotypes, and geometric mean titers against DENV1-4 were respectively 129, 216, 169, and 146. Six serious adverse events (SAEs), unrelated to vaccination, were reported including 2 virologically-confirmed dengue cases after the second vaccination in the control group. Reactogenicity of CYD-TDV decreased after each vaccination, was slightly higher than placebo, but not higher than either active control. Conclusions: Safety and reactogenicity of CYD-TDV were satisfactory and consistent with results from phase I and other phase II studies. Three doses of CYD-TDV induced a balanced neutralizing antibody response against the four dengue serotypes in children and adults living in a dengue endemic country. Citation: Tran NH, Luong CQ, Vu TQH, Forrat R, Lang J, et al. (2012) Safety and Immunogenicity of Recombinant, Live Attenuated Tetravalent Dengue Vaccine (CYDTDV) in Healthy Vietnamese Adults and Children. J Vaccines Vaccin 3:162. doi:10.4172/2157-7560.1000162


Introduction
In recent decades dengue has expanded in tropical and subtropical areas around the world to become one of the most important vectorborne diseases of humans, with around 2.5 billion people living in regions where the disease is transmitted [1]. The overall annual burden of dengue as reported by World Health Organization (WHO) is an estimated 50-100 million dengue infections and 500,000 hospitalizations for severe form of the disease [1]. This burden is projected to continue to increase, notably in association with the geographic expansion of the mosquito vectors (i.e., Aedes aegypti and Aedes albopictus) [2].
It is a high public priority to develop a vaccine against dengue, specifically for highly endemic areas such as the Mekong delta, considering that no specific treatment is available, and considering the high cost and challenges around vector control programs to sustainably control disease [3]. The impact of dengue on patients and their families is important in terms of economics and quality of life, the health burden is considerable for endemic regions [4].
Several candidate vaccines against dengue are in clinical or preclinical development, one of which is currently being evaluated in clinical efficacy trials in Asia and Latin America [5]. This vaccine -the CYD tetravalent dengue vaccine has been developed in compliance with WHO guidelines for the production and quality control of candidate tetravalent dengue vaccine [6], guidelines for the clinical evaluation of dengue vaccines in endemic areas [7], and guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses [8].
This vaccine contains four recombinant, live, attenuated viruses (CYD-1-4) developed based on the attenuated yellow fever vaccine virus, YF-17D, from which the genes encoding for pre-membrane and envelope proteins were removed and replaced with those of one of the four wild-type dengue viruses, one per serotype [5]. Phase 1 and early phase 2 trials have shown that a three-dose regimen of CYD-TDV elicits a balanced neutralizing antibody response against all four serotypes in flavi virus-naïve populations, as well as in populations previously exposed to dengue. The safety and reactogenicity profile observed in these studies were considered to be promising, and provided reassurance to progress to larger scale trials [5].
Viet Nam has had amongst the highest dengue disease burden in the Asia Pacific region, with an estimated 1 million dengue cases and more than 2000 deaths during the period 1991-2004 [9]. The Mekong Delta area of Southern Viet Nam is particularly affected, with reported incidence rates among school-age children of 16.9-40.4 per 1000 person-years [10]. All four dengue serotypes have co-circulated in this area in recent years, usually with one serotype dominating at a given time [11,12]. This dominant serotype however changes periodically [9].
We report on the first clinical study of CYD-TDV in Viet Nam, conducted in an area where dengue is highly endemic. Dengue and Japanese encephalitis (JE) viruses are the primary vector-borne flaviviruses in the country, [13] and while JE vaccine is included in the national immunization program this had only been partially implemented in the South of Viet Nam at the time of the study.
Individuals aged 2-45 years and in good health based on medical history, normal physical examination and laboratory parameters were eligible for inclusion. The main exclusion criteria were: personal or family history of thymic pathology, thymectomy or myasthenia; HIV, hepatitis B or C seropositivity tested at screening; immunodeficiency or receipt of immunosuppressive treatment; systemic hypersensitivity to any vaccine component; history of life threatening reaction to the trial vaccines or to vaccine containing any of the same substances; family history of allergy; vaccination with the control vaccines within the previous 3 years; pregnancy or breast-feeding. Females of child-bearing potential were instructed to use an effective means of contraception for at least 4 weeks prior to the first vaccination, until at least 4 weeks after the last vaccination.
Group allocation was performed using an Interactive Voice Response System (IVRS) based on randomization lists generated with the permuted block method and with age stratification. The study was conducted in accordance with Declaration of Helsinki and International Conference of Harmonization (ICH) guidelines for Good Clinical Practice (GCP) and was approved by the Vietnamese Ministry of Health Biomedical Research Ethics Committee as well as Joint Research Ethics Committee from Thailand. Written informed consent was obtained from all participants aged 12 years or older, and from the parent/guardian of all participants aged 2-17 years. Participants aged 8-11 signed an assent form. If either the participant or their parent/ legal representative were not able to read and sign the informed consent or assent form, it was signed by an impartial witness.
The study site fulfilled criteria set out in WHO guidelines for clinical evaluation of dengue vaccines in endemic areas [7].

Safety and reactogenicity
Participants were kept under observation for 30 minutes after each vaccination and were followed-up for safety until 6 months after the last vaccination. Participants or parents were provided with a thermometer, a ruler and a diary card to record any adverse event (AE) following each vaccination. The following AEs were documented and graded using a pre-defined severity scale as reported elsewhere solicited injection site reactions within 7 days (i.e., pain, erythema and swelling), solicited systemic reactions within 14 days (i.e., fever, headache, malaise, myalgia, and asthenia) and unsolicited AEs up to 28 days after each vaccination. All serious adverse events (SAE) were documented up to 6 months after the last vaccination.

CYD-TDV vaccine viremia
Cases of fever with body temperature ≥ 38°C for ≥ 48 hours within 28 days after vaccination were assessed for CYD-TDV viremia by quantitative RT-PCR. Serology and viremia analyses were performed at the sponsor's global clinical immunology laboratory (dengue PRNT, RT-PCR; Swift water, PA) and at the Center for Vaccine Development (dengue ELISAs, JE PRNT; University of Mahidol, Thailand). All testing was performed under blind conditions.

Serology
In serum collected at screening, Japanese encephalitis virus neutralizing antibodies levels were assessed by a plaque reduction neutralization test with a 50% endpoint (PRNT50) as described elsewhere [13,14]. Neutralizing serum antibody responses against dengue were measured using PRNT50 before and 28 days after each vaccination. Briefly, serial two-fold dilutions of heat-inactivated serum were mixed with a constant challenge dose of the dengue vaccine's four parental dengue wild-type viruses (target 80 PFU per well for each serotype) and inoculated into wells of a 24-well plate of confluent Vero cells. The neutralizing antibody titer was calculated and expressed as the highest reciprocal serum dilution (l/dil) reducing the mean plaque count by 50% as compared with the mean virus plaque number obtained from the control wells. The lower limit of quantization (LLOQ) of the dengue PRNT50 was 10 (1/dil) and seropositivity rate expressed the percentage of subjects with seropositive samples.

Passive detection and diagnosis of dengue fever
In the event of a febrile episode with a body temperature of ≥ 38°C for ≥ 48 hours and suspected dengue, participants/parents were instructed to return to the study center for laboratory diagnosis using quantitative dengue RT-PCR, and by commercial ELISA kits: NS1Ag ELISA (Platelia™, Biorad Laboratories, Marnes-La-Coquette, France),

Statistical method
All the main analyses were descriptive. For the main parameters, 95% Confidence Intervals of point estimates were calculated using the normal approximation for quantitative data and exact binomial distribution with Clopper-Pearson method for proportions. The sample size was arbitrarily set to 120 subjects in the dengue group and 60 subjects in the control group. Antibody titers were summarized by group and age cohort before and after each vaccination in terms of geometric mean titer (GMT), number and percentage with titer ≥ 10 1/dil against each dengue serotype (seropositivity) and against at least one, two, three, or the four dengue serotypes. Analyses included all participants present at the first visit and having received at least one vaccination (i.e., safety analysis set [SAS] and full analysis set [FAS] with available results from blood taken after vaccination).

Demographics and flavivirus immune status
Between March and July 2009, we enrolled 180 participants ( Figure  1) and vaccinated them with a first injection of CYD-TDV (N=120) or control vaccine (N=60); 172(96%) completed the study up to 6 month after the last vaccination. There were 6 early withdrawals in the dengue vaccine group and 2 in the control vaccine group. There were no discontinuations for safety reasons and none were lost to follow-up.
There were slightly more males enrolled in the control group (58% males) than in the dengue vaccine group (48% males). The mean age, height, weight, and body mass index (BMI), were comparable between groups (Table 1). At baseline, a high proportion of 77% of participants (76% in the dengue vaccine group and 80% in the control vaccine group) were seropositive against dengue or JE, and this proportion increased with age (Table 1). Almost 40% of participants had a baseline neutralizing antibody response against at least one dengue serotype: 39% (47/120) in the dengue vaccine group, 38% (23/60) in the control group. Seropositivity rates against dengue serotypes 1,2,3, and 4 were, respectively: 25%, 18%, 53%, and 23% among 2-5 year-olds, 50%, 53%, 55%, and 43% among 6-11 year-olds, 60 %, 65%, 70%, and 56% among 12-17 year-olds, and 95%, 95%, 95%, and 85.0% among 18 Table 2 shows the safety overview after any vaccination, by age cohort. Overall and by age group, solicited reactions were less frequently observed in the dengue vaccine group than in the control group. This was due to the solicited injection site reactions, reported for 33% [95%CI: 24.2; 41.7] for the dengue vaccine group than and 80% [95%CI: 67.7; 89.2] in the control group. The reporting rates of solicited systemic reactions were similar between groups ( Table 2). After dengue vaccination, reporting rates of solicited reactions and unsolicited adverse events appeared lower in children (i.e., 2-5 yo and 6-11 yo) than in adolescents (i.e. 12-17 yo) or adults (18-45 yo). The reporting rates of unsolicited adverse events were similar between groups ( Table  2). No adverse event led to study discontinuation, and there were no vaccine-related serious adverse events (SAE) ( Table 2).

Safety and reactogenicity
Injection site reactions after dengue vaccination were of grade 1 or 2 severity and resolved spontaneously, while a few grade 3 erythema and swelling reactions were recorded among 6-11 year-olds in the control group (Table 3). Most solicited injection site reactions after dengue vaccination were of grade 1 severity, and appeared within 3 days (data not shown). A few grade 3 systemic reactions (fever among 2-5 yearolds, and headache and asthenia among 18-45 year-olds) were reported after dengue vaccination (Table 3).  • Solicited reaction: any adverse event (AE) pre-listed in the protocol that is automatically considered to be vaccine-related, and as such termed an • 'adverse reaction' (AR) • Solicited injection site reaction: any AR associated with the site of injection that is pre-listed in the protocol (pain, erythema, swelling) • Solicited systemic reaction: any AR not associated with the site of injection that is pre-listed in the protocol (fever, headache, malaise, myalgia, asthenia) • Unsolicited AE: any observed AE that is not a solicited injection site or solicited systemic reaction • Unsolicited AR: any unsolicited AE that is considered to be vaccine-related SAE: any untoward medical occurrence that at any dose results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, is a congenital anomaly/birth defect, or is an important medical event  2) and 15% (95%CI: 8.9; 22.8) after the third. Overall, after any vaccination, headache (35%) was the most frequently reported solicited systemic reaction in the dengue group followed by malaise (29%), fever (28%), myalgia (20%) and asthenia (13%) while in the control group, the following reactions were reported: headache (30%), fever (27%), malaise (23%), myalgia (23%) and asthenia (5%). A summary of safety results according to age group is shown in table 3.

Serious adverse events
There were six serious adverse events (SAEs) due to hospitalization, none of which were related to vaccination. Three occurred in the dengue group (3/120, 2.5%), and three occurred in the control group (3/60, 5%). The three SAEs in the dengue group were acute gastritis, acute Pharyngitis, and dengue hemorrhagic fever (DHF) grade 1 meeting the 1997 WHO case definition [15]. The three SAEs in the control group were three cases of dengue infection (2 cases of DHF grade 1 and 1 case of DHF grade 2).

Fever and symptomatic dengue cases
From the start of the study through the period of 6 months followup after the last vaccination, 7 clinically diagnosed dengue cases were reported, 4 in the dengue vaccine group and 3 in the control vaccine group. All patients recovered without sequela. Of the 7 suspected dengue cases, 4 cases were inpatient cases (3 in the control vaccine group, 1 in the dengue vaccine group) and 3 outpatient cases (all in the dengue vaccine group). The outpatient case in the dengue vaccine group occurred within 28 days after the second vaccination in an 8 year-old female with a concomitant diagnosis of tonsillitis. Dengue infection was not confirmed in virological or serological tests. As this case occurred within 28 days of a dengue vaccine, serum was tested for vaccine viremia and found negative. Neither of the 2 outpatient cases in the control group were confirmed as dengue by serological or virological testing, and tonsillitis or another viral infection were suspected in addition to dengue. In the control group, there were 3 dengue cases that required hospitalization (i.e., one virologically confirmed grade I DHF due to serotype 1, one virologically confirmed grade II DHF also due to serotype 1, and one serologically probable grade I DHF). In the dengue vaccine group, there was one hospitalized grade I DHF in an 18 year-old male subject that occurred 93 days after the first vaccination. Virological tests for dengue were negative, although anti-dengue IgM was detected in acute and convalescent samples. He was seropositive for both dengue and JE viruses at baseline and on Day 28 after the first vaccination, his neutralizing antibody levels against serotypes 1-4 were 106, 191, 86, and 169 respectively. Of the 7 suspected dengue cases, dengue infection was therefore virologically confirmed in 2 cases in the control group (N=60), and none in the dengue vaccine group (N=120).

Immunogenicity
Antibody responses increased after each of the three doses of dengue vaccines, CYD-TDV against each serotype for all four serotypes in all age cohorts (Figure 2). On Day 28 after the complete 3-dose CYD-TDV schedule, GMTs against serotypes 1-4 were respectively 129, 216, 169, and 146, 92% of vaccines were seropositive to all 4 dengue serotypes, compared with 36.2% in the control group (Table 4). Antibody responses after vaccination appeared to increase with increasing age with GMTs after the third CYD-TDV dose in the range 64.7-143 among 2-5 yearsold, 93.9-185 among 6-11 years-old, 135-334 among 12-17 years-old, and 375-825 among adults ( Figure 2). Antibody responses to CYD-TDV were higher among those who were flavivirus seropositive at baseline compared with those who were flavivirus seronegative (Figure 3). In the flavivirus positive subgroup, GMTs increased to values in the range 187-316 after 3 doses of CYD-TDV, at which point 97.7% were seropositive against all four serotypes. In those who were flavivirus negative, GMTs increased to values in the range 31.2-75.5, at which point 75% were seropositive against all four serotypes.

Discussion
This is the first clinical trial of a dengue vaccine candidate in   Viet Nam. The high and age-dependent dengue seropositivity levels observed at the start of the study confirm the high endemicity of the disease in Long Xuyen. Regardless of flavivirus serostatus at baseline, antibody responses against the four dengue serotypes increased after each vaccination to levels consistent to those observed in other studies with this vaccine candidate in Philippines and Thailand, where dengue is also endemic [16,17]. That neutralizing antibody levels after the third dengue vaccination were higher in individuals with pre-existing antibodies against dengue or JE compared to those who were seronegative, is suggestive of a role of pre-existing flavivirus antibodies on dengue vaccine-induced antibody responses. Similar to results reported from the Philippines, immune responses to dengue vaccination also varied with age [16]. Post-vaccination antibody titers were highest in adults, whereas a higher relative increase compared to baseline was seen in younger age groups. In Singapore, where dengue is also endemic, but at lower levels than in Viet Nam, immune responses to the CYD-TDV vaccine candidate were also seen to vary with age, but with lower GMTs than those observed in our study (range 43-100) [18]. A similar effect of pre-existing flaviviral immune responses on the antibody responses after dengue vaccination has been observed with pre-existing yellow fever antibody responses [19,20].
CYD-TDV was immunogenic in this cohort of 2-45 year-olds and elicited balanced immune response against all four serotypes, as observed previously [16,18]. However the exact relationship between antibody titers, as assessed by PRNT50, and protection against dengue is not known, and a phase IIb proof-of-concept efficacy study of CYD-TDV conducted in Ratchaburi, Thailand showed protection against dengue serotypes 1, 3, and 4, but not against the serotype 2 viruses circulating at the time, despite having elicited satisfactory PRNT50 antibody titers against all four serotypes at levels comparable to those observed in our study [17]. The safety profile of the dengue vaccine candidate in this study was good, with no immediate unsolicited AEs, withdrawals for safety reasons, vaccine-related SAEs, or any other safety issues. Overall reactogenicity appeared similar to that of two licensed control vaccines (i.e., Meningococcal Polysaccharide Vaccine A+C, and Typhoid Vi polysaccharide vaccine (Typhim Vi®) and the rate of solicited reactions was lower in the CYD-TDV group. Local and systemic reactions were mild to moderate and short-lived and injection site pain was the most frequently reported injection site reaction. Reactogenicity was lower after the second and third vaccinations, compared to that documented after the first, confirming the good tolerability of this vaccine candidate.
Seven cases of dengue were clinically-suspected, but only two cases, both in the control group, were virologically confirmed to be dengue. Other cases tested positive for anti-dengue IgM, but given the probable effect of CYD-TDV vaccination on IgM and IgG responses, any such cases occurring after dengue vaccination (one case in our study) cannot conclusively be considered as dengue. The investigator's reporting of a second clinical diagnosis in these suspected, but not virologicallyconfirmed cases, adds to the likelihood that these cases were not dengue. The significant discordance between clinical diagnosis and laboratory confirmation highlights the importance of laboratory diagnosis of suspected infections.
CYD-TDV is a live attenuated viral vaccine and viral replication is expected to occur. Previous studies have shown that after vaccination, low levels of the CYD viruses can be detected in serum in a minority of vaccines between 7-14 days after vaccinations, but this has not been associated with any safety issues [16,22]. Here, one suspected dengue case occurring within 28 days of a dengue vaccination was tested and found negative for dengue vaccine viremia.
In summary, this study conducted in an area at high risk for dengue infection showed that a 3 dose CYD-TDV regimen elicited balanced neutralizing antibody responses against all four dengue virus serotypes in a wide range of age group, with a satisfactory safety profile comparable to that observed in previous trials. These data support the continued clinical development of the CYD-TDV vaccine candidate.

Conflict of Interest
TNH was the study principal investigator, LCQ and VTQH were coinvestigators. They did not receive direct payment from the study sponsor for their contributions and declare no conflicts of interest. WTA and FR designed the study. WTA, LJ, VQD, and BA interpreted the study results and are Sanofi Pasteur employees except VQD at the time of manuscript finalization.

Financial Disclosure
The study was sponsored by Sanofi Pasteur.