Associations of human leukocyte antigen with neutralizing antibody titers in a tetravalent dengue vaccine phase 2 efficacy trial in Thailand.

The recombinant, live, attenuated, tetravalent dengue vaccine CYD-TDV has shown efficacy against all four dengue serotypes. In this exploratory study (CYD59, NCT02827162), we evaluated potential associations of host human leukocyte antigen (HLA) alleles with dengue antibody responses, CYD-TDV vaccine efficacy, and virologically-confirmed dengue (VCD) cases. Children 4-11 years old, who previously completed a phase 2b efficacy study of CYD-TDV in a single center in Thailand, were included in the study. Genotyping of HLA class I and II loci was performed by next-generation sequencing from DNA obtained from 335 saliva samples. Dengue neutralizing antibody titers (NAb) were assessed as a correlate of risk and protection. Regression analyses were used to assess associations between HLA alleles and NAb responses, vaccine efficacy, and dengue outcomes. Month 13 NAb log geometric mean titers (GMTs) were associated with decreased risk of VCD. In the vaccine group, HLA-DRB1*11 was significantly associated with higher NAb log GMT levels (beta: 0.76; p = 0.002, q = 0.13). Additionally, in the absence of vaccination, HLA associations were observed between the presence of DPB1*03:01 and increased NAb log GMT levels (beta: 1.24; p = 0.005, q = 0.17), and between DPB1*05:01 and reduced NAb log GMT levels (beta: -1.1; p = 0.001, q = 0.07). This study suggests associations of HLA alleles with NAb titers in the context of dengue outcomes. This study was registered with clinicaltrials.gov: NCT02827162.

Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection.

BACKGROUND: Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. METHODS: The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3-11 years in Ratchaburi Province, Thailand. RESULTS: The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. CONCLUSIONS: This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

Serodiagnosis of asymptomatic dengue infection.

Dengue virus (DENV) is a mosquito-transmitted virus that is expanding across the world. The incidence of dengue infection, especially severe disease, has been increasing. DENV consist of 4 serotypes of single stranded RNA viruses (D1-D4) in the genus Flavivirus, family Flaviviridae. Majority of dengue infections are asymptomatic cases, which cause difficulty in disease control and are important in dengue surveillance. There is still no gold standard to diagnose asymptomatic dengue infection. Plaque reduction neutralization test (PRNT) has been developed for many purposes such as immunological study, clinical study, vaccine trial and is currently the most sensitive and specific method for serological surveillance. However, PRNT shows some degree of cross reaction among different dengue serotypes especially secondary dengue infection cases and to other flaviviruses. Moreover, various modification since the beginning make PRNT lack of inter-laboratory standardization which is an important issue. This paper discusses the important of asymptomatic dengue infection and its diagnostic method.

Detection of dengue cases by serological testing in a dengue vaccine efficacy trial: Utility for efficacy evaluation and impact of future vaccine introduction.

BACKGROUND: Dengue diagnosis confirmation and surveillance are widely based on serological assays to detect anti-dengue IgM or IgG antibodies since such tests are affordable/user-friendly. The World Health Organization identified serological based diagnosis as a potential tool to define probable dengue cases in the context of vaccine trials, while acknowledging that this may have to be interpreted with caution. METHODS: In a phase IIb randomized, placebo-controlled trial assessing the efficacy of a tetravalent dengue vaccine (CYD-TDV) in Thai schoolchildren, case definition was based on virological confirmation by either serotype-specific RT-PCRs or by NS1-antigen ELISA (Clinicaltrials.gov NCT00842530). Here, we characterized suspected dengue cases using IgM and IgG ELISA to assess their utility in evaluating probable dengue cases in the context of vaccine efficacy trials, comparing virologically-confirmed and serologically diagnosed dengue in the vaccine and placebo groups. Serologically probable cases were defined as: (1) IgM positive acute- or convalescent-phase samples, or (2) IgG positive acute-phase sample and ≥4-fold IgG increase between acute and convalescent-phase samples. RESULTS: Serological diagnosis had good sensitivity (97.1%), but low specificity (85.1%) compared to virological confirmation. A high level of false positivity through serology diagnosis particularly in the 2 months post-vaccination was observed, and is most likely related to detection of the immune response to the dengue vaccine. This lack of specificity and bias with vaccination demonstrates the limitation of using IgM and IgG antibody responses to explore vaccine efficacy. CONCLUSION: Reliance on serological assessments would lead to a significant number of false positives during routine clinical practice and surveillance following the introduction of the dengue vaccine.

Plaque reduction neutralization antibody test does not accurately predict protection against dengue infection in Ratchaburi cohort, Thailand.

BACKGROUND: The plaque reduction neutralization test (PRNT) is currently the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies to dengue virus, and in assessing the immunogenicity of a dengue vaccine. However, the correlation between presence of dengue-neutralizing antibody and protection from infection is not absolute. FINDINGS: In a cohort study in Ratchaburi Province, Thailand, 48 subjects with serologically confirmed symptomatic dengue infection were tested for pre-existing dengue neutralizing antibody using PRNT. Nine subjects had quite high pre-existing PRNT50 titers (titer >90) to subsequent infecting dengue serotypes, but still had symptomatic infections. CONCLUSION: This report provides evidence that PRNT may not be a good test for predicting protection against subsequent dengue infection.

Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial.

BACKGROUND: Roughly half the world's population live in dengue-endemic countries, but no vaccine is licensed. We investigated the efficacy of a recombinant, live, attenuated tetravalent dengue vaccine. METHODS: In this observer-masked, randomised, controlled, monocentre, phase 2b, proof-of-concept trial, healthy Thai schoolchildren aged 4-11 years were randomly assigned (2:1) to receive three injections of dengue vaccine or control (rabies vaccine or placebo) at months 0, 6, and 12. Randomisation was by computer-generated permuted blocks of six and participants were assigned with an interactive response system. Participants were actively followed up until month 25. All acute febrile illnesses were investigated. Dengue viraemia was confirmed by serotype-specific RT-PCR and non-structural protein 1 ELISA. The primary objective was to assess protective efficacy against virologically confirmed, symptomatic dengue, irrespective of severity or serotype, occurring 1 month or longer after the third injection (per-protocol analysis). This trial is registered at ClinicalTrials.gov, NCT00842530. FINDINGS: 4002 participants were assigned to vaccine (n=2669) or control (n=1333). 3673 were included in the primary analysis (2452 vaccine, 1221 control). 134 cases of virologically confirmed dengue occurred during the study. Efficacy was 30·2% (95% CI -13·4 to 56·6), and differed by serotype. Dengue vaccine was well tolerated, with no safety signals after 2 years of follow-up after the first dose. INTERPRETATION: These data show for the first time that a safe vaccine against dengue is possible. Ongoing large-scale phase 3 studies in various epidemiological settings will provide pivotal data for the CYD dengue vaccine candidate. FUNDING: Sanofi Pasteur.

Dengue infection in children in Ratchaburi, Thailand: a cohort study. I. Epidemiology of symptomatic acute dengue infection in children, 2006-2009.

BACKGROUND: There is an urgent need to field test dengue vaccines to determine their role in the control of the disease. Our aims were to study dengue epidemiology and prepare the site for a dengue vaccine efficacy trial. METHODS AND FINDINGS: We performed a prospective cohort study of children in primary schools in central Thailand from 2006 through 2009. We assessed the epidemiology of dengue by active fever surveillance for acute febrile illness as detected by school absenteeism and telephone contact of parents, and dengue diagnostic testing. Dengue accounted for 394 (6.74%) of the 5,842 febrile cases identified in 2882, 3104, 2717 and 2312 student person-years over the four years, respectively. Dengue incidence was 1.77% in 2006, 3.58% in 2007, 5.74% in 2008 and 3.29% in 2009. Mean dengue incidence over the 4 years was 3.6%. Dengue virus (DENV) types were determined in 333 (84.5%) of positive specimens; DENV serotype 1 (DENV-1) was the most common (43%), followed by DENV-2 (29%), DENV-3 (20%) and DENV-4 (8%). Disease severity ranged from dengue hemorrhagic fever (DHF) in 42 (10.5%) cases, dengue fever (DF) in 142 (35.5%) cases and undifferentiated fever (UF) in 210 (52.5%) cases. All four DENV serotypes were involved in all disease severity. A majority of cases had secondary DENV infection, 95% in DHF, 88.7% in DF and 81.9% in UF. Two DHF (0.5%) cases had primary DENV-3 infection. CONCLUSION: The results illustrate the high incidence of dengue with all four DENV serotypes in primary school children, with approximately 50% of disease manifesting as mild clinical symptoms of UF, not meeting the 1997 WHO criteria for dengue. Severe disease (DHF) occurred in one tenth of cases. Data of this type are required for clinical trials to evaluate the efficacy of dengue vaccines in large scale clinical trials.

Dengue infection in children in Ratchaburi, Thailand: a cohort study. II. Clinical manifestations.

BACKGROUND: Dengue infection is one of the most important mosquito-borne diseases. More data regarding the disease burden and the prevalence of each clinical spectrum among symptomatic infections and the clinical manifestations are needed. This study aims to describe the incidence and clinical manifestations of symptomatic dengue infection in Thai children during 2006 through 2008. STUDY DESIGN: This study is a school-based prospective open cohort study with a 9,448 person-year follow-up in children aged 3-14 years. Active surveillance for febrile illnesses was done in the studied subjects. Subjects who had febrile illness were asked to visit the study hospital for clinical and laboratory evaluation, treatment, and serological tests for dengue infection. The clinical data from medical records, diary cards, and data collection forms were collected and analyzed. RESULTS: Dengue infections were the causes of 12.1% of febrile illnesses attending the hospital, including undifferentiated fever (UF) (49.8%), dengue fever (DF) (39.3%) and dengue hemorrhagic fever (DHF) (10.9%). Headache, anorexia, nausea/vomiting and myalgia were common symptoms occurring in more than half of the patients. The more severe dengue spectrum (i.e., DHF) had higher temperature, higher prevalence of nausea/vomiting, abdominal pain, rash, diarrhea, petechiae, hepatomegaly and lower platelet count. DHF cases also had significantly higher prevalence of anorexia, nausea/vomiting and abdominal pain during day 3-6 and diarrhea during day 4-6 of illness. The absence of nausea/vomiting, abdominal pain, diarrhea, petechiae, hepatomegaly and positive tourniquet test may predict non-DHF. CONCLUSION: Among symptomatic dengue infection, UF is most common followed by DF and DHF. Some clinical manifestations may be useful to predict the more severe disease (i.e., DHF). This study presents additional information in the clinical spectra of symptomatic dengue infection.

Decay and persistence of maternal dengue antibodies among infants in Bangkok.

Abstract. Maternal dengue antibodies are important in determining the optimal age of dengue vaccination, but no study has quantified the heterogeneity of antibody decay and persistence in infants. We used longitudinal regression methods and survival analysis to measure decay and persistence times of serotype-specific neutralizing antibodies in 139 infants in Bangkok. A biphasic decay pattern was found with half-life times of 24-29 days between birth and 3 months and 44-150 days after 3 months. Atypical decay rates were found in 17% of infants for dengue virus-1 and -4. Median persistence times of plaque reduction neutralization tests > 10 ranged from 6 to 9 months. Persistence times for individuals could not be predicted based on antibody values at birth. Vaccination against dengue before 12 months of age would be ineffective if maternal antibodies at plaque reduction neutralization test levels below 80 interfere with vaccine uptake. Projections of average antibody persistence based on values at birth should be avoided in studies on dengue pathogenesis in infants.

Dengue antibody in Thai children from maternally transferred antibody to acquired infection.

This prospective cohort study examined serotype-specific neutralizing serum antibodies to dengue in 55 maternal-newborn pairs, at 3, 6, 9, 12, 18, and 24 months, and again at 4 to 8 years of age. In all, 95% of the mothers had neutralizing antibody to at least one dengue serotype, and 92.7% of the maternally transferred antibody had disappeared in 12-month-old infants. In all, 31 infants had serologically confirmed dengue infection; 5 symptomatic and 29 inapparent infections were reported. The increasing trend of inapparent infections among the older age group warrants further study of the need for effective dengue vaccination beyond 2 years of age.

Dengue in Thailand and Cambodia: an assessment of the degree of underrecognized disease burden based on reported cases.

BACKGROUND: Disease incidence data are needed to guide decision-making for public health interventions. Although dengue is a reportable disease in Thailand and Cambodia, the degree that reported incidence underrecognizes true disease burden is unknown. We utilized dengue incidence calculated from laboratory-confirmed outpatient and inpatient cases in prospective cohort studies to estimate the magnitude of dengue underrecognition and to establish more accurate disease burden estimates for these countries. METHODS AND FINDINGS: Cohort studies were conducted among children aged <15 years by members of a dengue field site consortium over at least 2 dengue seasons. Age-group specific multiplication factors (MFs) were computed by comparing data from three cohort studies to national surveillance data in the same province and year. In Thailand, 14,627 person-years of prospective cohort data were obtained in two provinces and 14,493 person-years from one province in Cambodia. Average annual incidence of laboratory-confirmed dengue was 23/1,000 and 25/1,000 in Thailand, and 41/1,000 in Cambodia. Calculated MFs in these provinces varied by age-group and year (range 0.4-29). Average age-group specific MFs were then applied to country-level reporting data and indicated that in Thailand a median 229,886 (range 210,612-331,236) dengue cases occurred annually during 2003-2007 and a median 111,178 (range 80,452-357,135) cases occurred in Cambodia in children <15 years of age. Average underrecognition of total and inpatient dengue cases was 8.7 and 2.6-fold in Thailand, and 9.1 and 1.4-fold in Cambodia, respectively. During the high-incidence year 2007, >95,000 children in Thailand and >58,000 children in Cambodia were estimated to be hospitalized due to dengue. CONCLUSION: Calculating MFs by comparing prospective cohort study data to locally-reported national surveillance data is one approach to more accurately assess disease burden. These data indicate that although dengue is regularly reported in many countries, national surveillance data significantly underrecognize the true burden of disease.

Safety and immunogenicity of a single administration of live-attenuated Japanese encephalitis vaccine in previously primed 2- to 5-year-olds and naive 12- to 24-month-olds: multicenter randomized controlled trial.

BACKGROUND: Safe and effective Japanese encephalitis (JE) vaccines are needed to protect populations living in or visiting endemic areas. A live-attenuated JE-chimeric virus vaccine (JE-CV) has been developed with a single-dose regimen. METHODS: In an open-label, crossover study, 100 children aged 2 to 5 years with a history of 2-dose primary vaccination with mouse-brain derived inactivated JE vaccine according to the Thai Expanded Program for Immunization schedule, and 200 JE vaccination-naive 12- to 24-month-old toddlers were randomized 1:1 to receive JE-CV, containing ≥4 log10 plaque forming units, 1 month before or after hepatitis A control vaccine. Neutralizing antibody titers were assessed using PRNT50 (titers expressed in inverse of dilution) before and 28 days after JE-CV, and at months 7 and 12. RESULTS: All 2- to 5-year-olds and 96% of 12- to 24-month-olds were seroprotected (titer ≥10) 28 days after JE-CV administration, and geometric mean titers (GMT) (95% confidence interval) in these age groups were 2634 (1928-3600) and 281 (219-362), respectively. One year later, seroprotection rates in the 2 age groups were 97% and 84% and GMTs were 454 and 62.3, respectively. Vaccine-induced antibodies neutralized a panel of wild-type JE isolates. There were no vaccine-related serious adverse events. Reactogenicity of JE-CV was comparable with that of the inactivated hepatitis A vaccine. CONCLUSIONS: A single administration of JE-CV has a good safety profile and elicits a protective immune response in both JE-naive toddlers and JE-primed young children.

A three-year clinical study on immunogenicity, safety, and booster response of purified chick embryo cell rabies vaccine administered intramuscularly or intradermally to 12- to 18-month-old Thai children, concomitantly with Japanese encephalitis vaccine.

After concomitant administration of purified chick embryo cell rabies vaccine and Japanese encephalitis vaccine to toddlers, adequate rabies and Japanese encephalitis virus neutralizing antibodies concentrations were demonstrated by day 49, 7 days after a booster at 1 year, and in the majorly at 3 years postvaccination. The inclusion of rabies vaccine in the expanded program on immunization should be considered in rabies endemic countries.

Age-specific prevalence of dengue antibodies in Bangkok infants and children.

Seroprevalence of dengue antibodies were determined in healthy children in Bangkok. At 9, 12, and 18 months of age 23%, 9%, and 17% of children respectively had neutralizing antibodies against one or more serotypes. DEN1 was the most prevalent, followed by DEN3, DEN2, and DEN4. Twelve children had serologic evidence of dengue infection. The nadir of dengue antibodies in children was at 12 months of age. In highly endemic areas, dengue vaccination could be needed at an early age.

Comparative study of the effectiveness and pharmacokinetics of two rectal artesunate/oral mefloquine combination regimens for the treatment of uncomplicated childhood falciparum malaria.

BACKGROUND: Rectal artesunate has been shown to be an effective treatment for falciparum malaria and is useful in patients who cannot take medicine orally or when parenteral medication is inconvenient. A combination with mefloquine can decrease the duration of treatment, increase compliance and delay development of resistance. There are no clear data on whether a higher dosage of rectal artesunate results in a better clinical response. AIM: To assess two rectal artesunate/oral mefloquine regimens for treating uncomplicated multi-drug-resistant childhood falciparum malaria. METHODS: Seventy children aged 1-14 years with uncomplicated falciparum malaria were randomly assigned to receive either 10 (range 8-12) or 20 (range 16-24) mg/kg/day rectal artesunate for 3 days followed by 25 mg/kg oral mefloquine. The study endpoints were fever clearance time, parasite clearance time and proportion of patients with recrudescence. Serum levels of artesunate and dihydro-artemisinin were measured after the first dose of rectal artesunate in 16 subjects. RESULTS: Both regimens were safe and effective. The cure rate was 100% in the 53 patients who completed 28-day follow-up. All of the study endpoints were comparable between both treatment groups. CONCLUSION: A regimen of rectal artesunate 10 mg/kg/day for 3 days followed by mefloquine 25 mg/kg is optimal for the treatment of uncomplicated falciparum malaria. There was no definite benefit from increasing the dosage of rectal artesunate from 10 to 20 mg/kg/day.

Scientific consultation on immunological correlates of protection induced by dengue vaccines report from a meeting held at the World Health Organization 17-18 November 2005.

Several dengue vaccine candidates have been evaluated in early clinical phase, and some are scheduled for efficacy testing in population-based studies. Given the advancements in dengue vaccine development, there is an increased interest in identifying immunological correlates of protection for these vaccines in order to facilitate their evaluation, further refinement, production and registration. To this end, the WHO Initiative for Vaccine Research (IVR) convened a consultation on primary and secondary immunological correlates of protection induced by dengue vaccines. The meeting was held on the 17th and 18th of November, 2005 at WHO headquarters in Geneva. The consultation was a first dedicated review of the available data in support of establishing correlates. It is concluded that it is not yet possible to define one specific set of correlates, the consultation concluded in recommendations that should help to gather the missing evidence in conjunction with future vaccine trials.

Dengue virus infections in the first 2 years of life and the kinetics of transplacentally transferred dengue neutralizing antibodies in thai children.

BACKGROUND: Understanding dengue virus infection in children and the kinetics of maternal dengue neutralizing antibodies is essential for effective dengue immunization of children in endemic areas. METHODS: Serum samples from 219 mother-child pairs and 140 children at 3, 6, 9, 12, 18, and 24 months of age from Bangkok, Thailand, were tested for serotype-specific dengue antibodies. Febrile episodes in the children were recorded. RESULTS: Antibodies were found in 97% of cord serum samples and disappeared in 27%, 80%, and 95% of the children by the age of 6, 9, and 12 months, respectively. Geometric mean titers (GMTs) of the antibodies to 4 dengue serotypes decreased to 5.4-15.5 in 6-month-old infants. Eleven of 12 children acquired dengue virus infection at 6 months of age and beyond; 1 had the infection at 3 months of age. Two exhibited undifferentiated febrile illnesses, and 10 had subclinical infections. CONCLUSIONS: Evidence of dengue virus infection and very low GMTs against all dengue serotypes in children at 6 months of age and beyond was demonstrated. There was no evidence that maternal antibodies were harmful to infants. Dengue virus infection rates increase from 12 months of age onward. These data provide information for supporting the optimal age at vaccination.

Short report: immune response and occurrence of dengue infection in thai children three to eight years after vaccination with live attenuated tetravalent dengue vaccine.

From 1992 to 1997, 140 Thai children 4-15 years of age received an investigational live attenuated tetravalent dengue vaccine (LATDV). These children were contacted 3-8 years later in 2001 to assess humoral immunity and investigate whether they were subsequently at higher risk of developing severe dengue. One hundred thirteen were successfully contacted and participated in this retrospective cohort study with two age- and address-matched controls per vaccinee. The number of vaccinated subjects with neutralizing antibodies increased compared with 3-8 years earlier, which was probably due to subsequent wild-type dengue infections. There were no excess hospitalizations for clinically suspected dengue fever (DF) or dengue hemorrhagic fever (DHF) in vaccinees (one with DF and three with DHF) compared with controls (14 with DHF). Results suggest that preexisting dengue antibodies induced by LATDV do not enhance dengue illness, and the use of the vaccine in a dengue-endemic area is safe.

Life-saving rectal artesunate for complicated malaria in children.

We report the effectiveness of two regimens of rectal artesunate formulation in treating 13 Thai children with cerebral/complicated falciparum malaria. The drug was given at an initial dose of 40 mg/kg bodyweight, in 3 or 4 divided doses in the first 24 hours, followed by 10 mg/kg bodyweight once daily for three consecutive days. Mefloquine, at a dose of 15 mg/kg bodyweight was given orally at 72 hours after the initial dose of artesunate, followed by 10 mg/kg bodyweight 6 hours later. Three cases with cerebral malaria gained consciousness within 20 hours of artesunate administration. The median time required for reduction of parasitemia by 90% of the initial value (P90) in 13 children was 11.2 hours. No recrudescence was observed in any of the patients during the 28-day follow-up period. Plasma concentrations of artesunate and dihydroartemisinin (active plasma metabolite of artesunate) measured in two patients who received the high initial dose regimen (20 mg/ kg bodyweight) suggested rapid absorption and adequate plasma concentrations of both compounds following the administration of artesunate via the rectal route. Further studies for the optimized regimen of rectal artesunate in the treatment of cerebral/complicated childhood falciparum malaria in areas of multidrug resistance are warranted.