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Dengue infection and advances in dengue vaccines for children
Review
Dengue infection and advances in dengue vaccines for children Scott B Halstead, Leonila F Dans
Dengue viruses are endemic in most tropical and subtropical countries where they produce disease ranging from a mild fever to a severe, potentially fatal vascular permeability syndrome. We reviewed the status of development and testing in children of three vaccines designed to protect against the four dengue viruses. The first dengue virus vaccine, Dengvaxia, now licensed in 20 endemic countries, the EU and the USA, provides protection against severe dengue in seropositive individuals but increases the risk for naive recipients to develop severe dengue and to be hospitalised. We discuss mechanisms and implications of shortcomings of the licensed vaccine and describe the structure and attributes of two other dengue virus vaccines. Based upon human dengue challenge studies, one of these vaccines promises to deliver solid, long-lasting immunity after a single dose. Because dengue virus infections are ubiquitous in residents and visitors to tropical countries, in the absence of a protective vaccine paediatricians should recognise the early signs and clinical presentation of severe dengue, understand its pathophysiology and appropriate management.
Introduction There are four closely related dengue viruses (designated DENV-1, 2, 3, and 4). All dengue viruses cause an acute febrile disease and are transmitted by the bite of Aedes aegypti, an anthropophilic mosquito that infests virtually all tropical and subtropical countries.1 Hundreds of millions of people are infected every year with outcomes that are variable and unique (figure).3,4 From the perspective of human health, the most important feature of dengue is the ability of a first infection, or passively acquired dengue antibodies, to increase the severity of the next dengue virus infection.5 This immunopathological phenomenon, termed anti body dependent enhancement (ADE), is coupled with a late-in-illness vascular permeability.6 Vascular permeability during dengue infections, identified as the dengue vascular permeability syndrome, allows fluid and small macromolecules to escape circulation and is accompanied by thrombocytopenia, altered haemostasis, elevated liver enzymes, elevated concentrations of cytokines or chemokines and activation of complement.6–8 Compelling evidence exists to suggest that much of this damage is produced by circulation during the febrile phase of a toxic viral non-structural protein 1 (NS1).9,10 For reasons not fully understood, not all humans are equally at risk to this immunopathological outcome. Attack rates are substantially lower among dengue virus-infected subSaharan Africans than among European or Asian populations.11 Moreover, the syndrome is most severe in young children, the elderly, and those with pre-existing conditions.12,13 Despite the fact that A. aegypti was largely eradicated from the Americas during the successful effort to conquer urban yellow fever in the early 20th century, sustained control of this mosquito has proved impossible.14 Dengue is a major human infectious disease in endemic countries and a substantial cause of febrile disease in tourists to tropical destinations.15 As a result there is a major effort to develop protective vaccines. However, the success of vaccines has been impacted by the dengue immuno pathology phenomenon. This Viewpoint looks at the status of vaccine development, testing, and introduction
in the context of what is known about disease causation and innate clinical responses to dengue infection.
Dengue immunopathology
Lancet Child Adolesc Health 2019 Published Online August 1, 2019 http://dx.doi.org/10.1016/ S2352-4642(19)30205-6 Uniformed Services University of the Health Sciences, Bethesda, MD, USA (Emeritus Prof S B Halstead MD); and Departments of Pediatrics and Clinical Epidemiology, College of Medicine, University of the Philippines, Manila, Philippines (Prof L F Dans MD) Correspondence to: Prof Scott B Halstead, Rockville, MD 20852, USA [email protected]
Until the 1950s, dengue was known as a disease that produced epidemics of self-limited febrile exanthemata with little mortality. This pattern changed when shock and gastrointestinal haemorrhage started being reported in association with dengue infection.16 That severe dengue might be an immunological phenomenon was suggested when it was discovered that dengue haemorrhagic fever and dengue shock syndrome accompanied a second heterotypic dengue virus infection.17 Investigating this phenomenon in rhesus monkeys, the rates of viraemia were higher during second infection than first infections with the same dengue virus.18 In addition, dengue virus grew more abundantly in cultures of peripheral blood Key messages • Three dengue vaccines for children are in or have completed phase 3 clinical testing. • Two of these vaccines are molecular chimeras, splicing structural genes of the four dengue viruses into the non-structural genes of yellow fever vaccine virus (Dengvaxia [Sanofi Pasteur]) or DENV-1, 3, and 4 genes into the DENV-2 vaccine virus (TAK-003 [Takeda Pharmaceuticals]). • The third vaccine, live-attenuated tetravalent dengue vaccine (US National Institutes of Health, NIH), contains mutagenized DENV-1, 3, and 4 and a DENV-4-and-2 chimera. A single dose solidly protected seronegative humans from recombinant non-parental DENV-2/3 challenge in phase 2b clinical trials. • In a phase 3 trial in ten dengue-endemic countries Dengvaxia provided protection against severe dengue in dengue virus seropositive children but sensitised seronegative children for risk of enhanced disease during breakthrough infections. • Dengue virus NS1 circulating at high concentrations during dengue virus infections is directly toxic to endothelial cells. • Dengvaxia contains no dengue virus NS1s while the NIH dengue vaccine contains DENV-1, 3, and 4 non-structural 1 proteins. Clinical efficacy trial results suggest that dengue virus non-structural 1 protein immunity might be important for protection. • Until dengue has been controlled paediatricians should have the knowledge to accurately diagnose acute dengue, recognise warning signs of severe dengue, and be prepared to provide life-saving resuscitative treatment.
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<10 10–99 100–199 200–499 500–999 1000–1499 1500–2999 ≥3000
West Africa
Caribbean
ATG
VCT
Barbados
Comoros
Dominica
Grenada
Maldives
Mauritius
LCA
TTO
TLS
Seychelles
Eastern Mediterranean
Marshall Isl
Kiribati
Solomon Isl
FSM
Vanuatu
Samoa
Fiji
Tonga
Malta
Persian Gulf
Singapore
Balkan peninsula
Figure: Age-standardised incidence rates of dengue (per 100 000 person-years), in 2013 ATG=Antigua and Barbuda. VCT=St Vincent and Grenadines. Isl=Island. FSM=Federated States of Micronesia. LCA=St Lucia. TTO=Trinidad and Tobago. TLS=Timor Leste. Adapted from Stanaway and colleagues, 2016.2
monocytes from dengue virus-immune humans than in non-immune humans. Furthermore, enhanced dengue virus infections could be achieved regularly when IgG dengue virus antibodies were added to dengue virus in cultures of primary human monocytes.19,20 This phenomenon extends to other acute and chronic human and animal infections in which cross-linking of IgGmicrobe immune complexes with Fcγ receptors results in increased intracellular infection contributing to enhanced disease severity, a mechanism labelled intrinsic ADE (iADE).21,22 iADE differ from extrinsic ADE (eADE), a cell surface phenomenon resulting from a more efficient attachment of immune complexes to Fc receptors than the virus itself attaching to viral receptors. eADE contributes a three-fold increase while iADE a 100-fold increase in virus production.23 Maternal dengue virus antibodies might protect newborns from dengue virus infection for several months, followed by several weeks of months of increased risk to dengue vascular permeability syndrome. This ADE event is common in the highly endemic countries of southeast Asia where mothers experience two or more lifetime dengue virus infections.20 Infant dengue vascular permeability syndrome-ADE during a first dengue virus infection, fortunately, is unique in human medicine.24–27 2
Dengue disease causation Late in the febrile period of dengue vascular permeability syndrome there might be a sudden and profound capillary leak. At first it was thought that when an increased number of dengue virus-infected target cells (monocytes, macrophages and dendritic cells) was attacked by the immune elimination response, a marked release of pro-inflammatory and anti-inflammatory factors resulted.28 These factors were thought to damage capillary integrity resulting in hypovolemic shock (also called a cytokine storm).29 The cytokine storm hypothesis has now been replaced by evidence of the direct pathogenicity of dengue virus NS1. NS1 is produced during cellular infections with each of the four dengue viruses.9 Instead of remaining cell-bound, dengue NS1 circulates in great quantities as a hexamer in blood during acute phase.30 For several decades it has been known that immunisation of laboratory animals with dengue virus NS1 protected them against lethal dengue virus challenges31 and that concentrations of NS1 in serum during an early acute phase correlated directly to the ensuing disease severity during secondary dengue virus infections.32 In 2015, a molecular analogy was observed between bacterial lipopolysaccharides and that of dengue virus NS1.33 Both bacterial lipopolysaccharides
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and NS1 interact with Toll-like receptor 4 on the surface of monocytes, macrophages, and endothelial cells to release a range of cytokines and chemokines. These same mediators are detected in blood from patients with dengue vascular permeability syndrome during the acute phase. In vitro, NS1 resulted in the disruption of the integrity of endothelial cell monolayers. The authors concluded that dengue vascular permeability syndrome might be a viral protein toxicosis. NS1-mediated cyto kine release could be inhibited by the Toll-like receptor 4 antagonist lipopolysaccharides-Rhodobacter sphaeroides, suggesting an avenue for therapeutic intervention. The same observation was confirmed in an in vivo model.34 In addition, DENV-2 NS1 inoculated intra venously at physiologically relevant concentrations in sub-lethally DENV-2-infected C57BL/6 mice produced lethal vascular permeability.35 Vaccination of mice with DENV-2 NS1 protected them against endothelial leak age and death from lethal DENV-2 challenge. Mice immunised with all four dengue virus NS1 proteins were completely protected against homologous dengue virus challenges while immunisation with DENV-1 NS1 partially protected against heterologous DENV-2 challenge. In other experiments, dengue virus NS1 was shown to directly alter the barrier function of pulmonary endothelial cell mono layers through disruption of the endothelial glycocalyx-like layer by triggering the activation of endothelial sialidases, cathepsin L, and heparanase, which are enzymes responsible for degrading sialic acid and heparan sulphate proteo glycans.36 Separately, disruption of endothelial glyco calyx components had been shown to correlate with plasma leakage during severe dengue virus infection in humans.37,38 The contribution of these dengue virus NS1-induced endothelial cell intrinsic pathways was found to be independent of inflammatory cytokines but dependent on the integrity of endothelial glycocalyx components both in vitro and in vivo.35 In conclusion, NS1 toxicosis is demonstrably an efferent mechanism of vascular permeability in mice directly related to intra cellular dengue virus production that can be modified by ADE. Based upon the kinetics of human dengue virus infections, a role for NS1 in endothelial damage, complement activation, liver damage, and haemostatic abnormalities is plausible.10
Innate host responses to dengue virus infection Infection in the absence of dengue virus antibodies
In infants and children aged 1–5 years, a first dengue virus infection is commonly inapparent.39 During midchildhood, an initial dengue virus infection usually results in a short febrile disease, the dengue fever syndrome, with potential headache, flushed face, inappetence, upper respiratory symptoms, nausea, vomiting, myalgia, leukopenia, and prostration.39 First dengue virus infections in adolescents are generally more reactogenic leading to school absenteeism in
10–20% of such individuals and some hospitalisations.40 Most non-indigenous children who acquire a first dengue virus infection while traveling or residing abroad develop this syndrome.
Infection in the presence of dengue virus antibodies Dengue virus infection of infants circulating passively acquired dengue antibodies from mothers, who have had multiple dengue virus infections, or young children (1–8 years of age) immune to a single prior dengue virus infection might initially produce mild dengue fever-like symptoms. After 2–3 days of fever, these symptoms can evolve to the signs and symptoms of dengue vascular permeability syndrome, including sudden weakness, anorexia, lethargy, mid-epigastric abdominal pain, and persistent vomiting. More alarming findings are cool extremities, scattered petechiae, thrombocytopenia (<100 000 mm³), anuria, a weak and rapid pulse, liver aspartate aminotransferase or alanine aminotransferase concentrations of 1000 (IU/L) or greater, circumoral cyanosis, an increased haematocrit with concurrent decrease in platelet count, hypovolaemia, low serum albumin, and sonographic evidence of serosal effusions. Those clinical findings that precede shock have been identified as warning signs.41 During the febrile period the signs and symptoms of dengue vascular permeability syndrome might change from day to day. Major changes in blood pressure occur as dengue vascular permeability syndrome progresses, including increased peripheral vascular resistance with decreased cardiac output and normal or low central venous pressure.42 Shock is not due to congestive heart failure but to venous pooling. With increasing cardiovascular compromise, the diastolic pressure rises toward the systolic and the pulse pressure narrows. Finally, there is decompensation and both pressures disappear abruptly. Because of increased peripheral vascular resistance, shock in dengue patients frequently manifests as a narrow pulse pressure with normal systolic values. To the unwary, dengue shock might not be recognisable. Before the modern era of proactive and careful fluid resuscitation, gastrointestinal bleeding occurred frequently in shocked children. During hypotension, blood is shunted from the splanchnic to the cerebral vascular system resulting in tissue anoxia in the gastrointestinal tract. On autopsy, shock-related gastrointestinal bleeding is by diapedesis.43 Children might be more susceptible to vascular permeability shock-related gastrointestinal bleeding than adults because of age-related differences in the integrity of vascular endothelial barriers.12,44,45 Dengue vascular permeability syndrome is unstable in the youngest patients. As children approach adolescence, the frequency of dengue vascular permeability syndrome and case fatality rate decrease. The innate susceptibility of younger vaccinated children compared with older vaccinated children (to dengue vascular permeability syndrome accompanying
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breakthrough dengue virus infections) came as a surprise to phase 3 clinical trial observers.46,47 However, the phenomenon had been described during the DENV-2 epidemic in Havana, Cuba, in 1981, where 4 years earlier 45% of the population had been infected with DENV-1.12
Disease management
For the EU license see https:// globenewswire.com/newsrelease/2018/12/19/1669374/0/ en/Dengvaxia-vaccineapproved-for-prevention-ofdengue-in-Europe.html For the US FDA license see https://www.fda.gov/newsevents/press-announcements/ first-fda-approved-vaccineprevention-dengue-diseaseendemic-regions
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Because of the high volume of travel to tropical destinations, patients with acute dengue might present to any clinical practice at any time of the year, so paediatricians should always be prepared.41,48 Success ful management of dengue vascular permeability syndrome relies on meticulous regulation of parenteral fluids and colloids during the period of increased vascular leakage, together with proactive management of major bleeding.41 The physician should remember that all fluid administered will be reabsorbed and fluid overload might result. Fluid overload is believed to contribute a substantial proportion of all deaths resulting from dengue vascular permeability syndrome.48 Because the fluid lost is approximately equivalent to plasma, isotonic crystalloid solutions are recommended, except in very young infants (<6 months of age), in whom 0·45% sodium chloride can be used. From a double-blind randomised comparison of three fluids for initial resuscitation of 383 Vietnamese children with dengue shock, it was demonstrated that Ringer’s lactate is sufficient to resuscitate children with moderately severe dengue shock syndrome.49 In patients with massive plasma leakage and those who do not respond to the maximum volume of crystalloid, hyperoncotic colloid solutions, such as 10% dextran 40 in saline, are used. Iso-oncotic colloids or albumin are not recommended. Except in patients with shock, the volume of fluid administration in patients with plasma leakage is limited to maintenance fluid requirements plus 5% deficit (5% of body weight) to account for dehydration; if patients are taking oral fluids, the recommended volume reflects the total of oral and intravenous fluids.48 The coagulopathy associated with dengue infections is well described, but unfortunately the underlying mechanisms remain unclear. Severe bleeding occurs only rarely in children (almost invariably in association with profound shock), and thrombotic complications are not seen. An increase in activated partial thrombo plastin time and a reduction in fibrinogen concentrations are fairly consistent findings, together with thrombo cytopenia, and these abnormalities tend to correlate with overall severity.50 The evidence for classical dis seminated intravascular coagulation in most cases is not convincing.50 Thrombocytopenia is almost invariable in patients with dengue, with several mechanisms thought to be involved. Early bone marrow suppression combined with increased peripheral destruction of platelets
during the febrile and early convalescent phase of the disease can lead to profound thrombocytopenia with platelet count nadirs as low as 5000 per mm³ for some cases.51 However, during the recovery period platelet numbers rise promptly as production increases in the hyper-cellular marrow. Platelet transfusions are widely administered in the mistaken belief that they will prevent severe bleeding. Many physicians treat the name of the disease (dengue haemorrhagic fever) rather than the patient. In the absence of profound bleeding (ie, bleeding sufficient to warrant consideration of blood transfusion) there is no evidence that prophylactic platelet transfusions improve outcome but offer a very definite risk both of acute and longterm complications.51
Vaccines
Dengvaxia Dengvaxia, developed by Sanofi Pasteur, is the only licensed dengue vaccine. It is live-attenuated tetravalent chimeric vaccine that incorporates the structural genes of the four dengue viruses into the genome of the yellow fever vaccine. It has been tested for vaccine efficacy and safety in placebo-controlled clinical trials enrolling approximately 35 000 children, aged 2–16 years in ten dengue-endemic countries.46 Efficacy results, published in 2015, were mixed. At year 3 after the first dose, vaccine protection of 9–16-year-old children against hospitalisation was 65·5% whereas for children aged 8 years or younger the rate was 44·6%.46 In the original phase 3 report, the serostatus at the time of vaccination was known only for a small sample. Among seropositive individuals, in the age group 2–8 years, vaccine efficacy was 70·1%, while among 9–16 year olds, efficacy was 81·9%. However, outcomes were quite different in seronegative children. In 2–8 year olds protection was 14·4% whereas for 9 years and older, it was 52·5%.46 More pointedly, among 2029 vaccinated children of 5 years or younger, 20 (0·99%) were hospitalised for dengue, a substantially higher proportion than controls, in whom the proportion was two (0·2%) per 1005, a relative risk of 4·95 (p=0·03). These data led the manufacturer to recommend the vaccine be restricted to individuals of 9 years of age and older.46 Despite the evidence that Dengvaxia was asymmetrically protective and enhancing in young children, WHO’s Scientific Advisory Group of Experts (SAGE) endorsed vaccine use for children of 9 years of age and older, followed by licensing of the product in 20 dengue-endemic countries.47,52 The EU and the US FDA issued licenses for Dengvaxia to be given to dengue-seropositive individuals resident in dengue-endemic areas. However, the serological test or tests that specifically and reliably identify dengue sero positive individuals were not identified. In 2016, SAGE recommended that Dengvaxia be given to individuals of 9 years or older residing in populations, national or subnational, with a seroprevalence of 70%
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or greater.53 To provide guidance for the deployment of Dengvaxia in high endemicity populations, WHO described case sampling and statistical methods to identify areas of high endemicity where dengue virus seroprevalence could be measured using recommended designs for sampling.54,55 To evaluate the safety of giving Dengvaxia to 9–16 year olds irrespective of sero logical status at time of vaccination, Sanofi sponsored develop ment of a serological test capable of distinguishing antibodies derived from Dengvaxia from those raised by wild-type dengue virus infections.56 This test was applied to sera from a cohort representing 10% of children taking part to a phase 3 randomized controlled trial, for all of whom blood samples had been collected during month 13 post vaccination.57 This study established that dengue seronegativity, rather than age at vaccination, placed children at a significantly increased risk of acquiring severe hospitalised dengue disease 1 year or more after vaccination compared with age and serostatus-matched controls.57 Based upon these new data, SAGE, the Global Advisory Committee on Vaccine Safety, and the WHO Dengue Vaccine Working Group reconsidered their 2016 recommendations and issued the following summary statement: “For countries considering vaccination as part of their dengue control programme, pre-vaccination screening is the recommended strategy. With this strategy, only persons with evidence of a past dengue infection would be vaccinated (based on an antibody test, or on a documented laboratory confirmed dengue infection in the past). If pre-vaccination screening is not feasible, vaccination without individual screening could be considered in areas with recent documentation of seroprevalence rates of at least 80% by age 9 years”.58
TAK-003 In January, 2018, Takeda Pharmaceuticals announced the completion of phase 3 trials for their vaccine, TAK-003.59–62 Efficacy and safety data for this vaccine have not yet been published. This vaccine consists of a live-attenuated DENV-2 strain and three chimeric viruses containing the prM and E protein genes of DENV-1, 3, and 4 expressed on the backbone of the DENV-2 genome.63,64 These chimeric dengue viruses have a long history. The parental DENV-1 and 2 from Thailand, DENV-3 from the Philippines, and DENV-4 from Indonesia incorporated into the TAK-003 vaccine were provided to the US Centers for Disease Control and Prevention Vector-Borne Diseases Branch, Fort Collins from the University of Hawaii.65 There, in 1980, DENV-1, 2, and 4 had been adapted to primary dog kidney and DENV-3 to African green monkey kidney then transferred to Mahidol University, Bangkok, where they were further passaged, tested for virulence markers and shown to be attenuated and immunogenic at different passage rates in susceptible adult volunteers.65,66 The attenuated viruses were licensed to Sanofi-Aventis, tested in thousands of volunteers of all ages but ultimately abandoned due to under-attenuation of the DENV-3 strain
serially passaged in African green monkey kidney.67 Of these four viruses, the most successful vaccine candidate was DENV-2 16881 primary dog kidney 53, which produced exceptionally high rates of seroconversions in seronegative human volunteers who had minimal dengue signs or symptoms.68 The attenuating mutations were identified and an infectious cDNA clone constructed.69,70 This DENV-2 vaccine is one component and the backbone for DENV-1, 3, and 4 in the TAK-003 vaccine. The developers hope for successful protection against all dengue virus infection or disease based on the broad neutralizing antibody responses that follow two doses of TAK-003. Clinical data from phase 3 clinical trials are awaited.
Live-attenuated tetravalent dengue vaccine For nearly 20 years, staff at the National Institute of Allergy and Infectious Diseases in Bethesda (MD, USA) and the Johns Hopkins Bloomberg School of Public Health in Baltimore (MD, USA) have been designing and testing dengue vaccine candidates. Some dengue viruses were attenuated by removing nucleotides from the non-translated region of the dengue genome (Δ30). A crucial component of this development programme was that monovalent vaccine candidates were tested for immunogenicity and attenuation in seronegative human volunteers.71 The final product, live-attenuated tetravalent dengue vaccine, contains genetically attenuated DENV-1, 3, and 4 viruses, and a fourth component, a chimera of structural genes of DENV-2 inserted into DENV-4.72–74 Having received a single dose of live-attenuated tetravalent dengue vaccine, volunteers were solidly protected from viraemia, dengue rash, or anamnestic antibody response following challenge with live-attenuated DENV-2 Δ30 (Tonga 74 strain) or DENV-3 Δ30 (Sleman 78 strain), recovered from mild dengue outbreaks and having major genetic differences from parental dengue virus (Durbin A, John Hopkins Bloomberg School of Public Health, personal communication).75 That this protection is likely to be of long duration is evidenced by the solid immune response observed to a booster dose of live-attenuated vaccine given 12 months after initial dose.76 These results are complemented by evidence that a single dose of liveattenuated tetravalent dengue vaccine raises monospecific DENV-1–4 neutralizing antibodies conformationally similar to those after human infections with wild-type dengue viruses and that correlate with protection.77,78 Moreover, the T cell responses to live-attenuated tetravalent dengue vaccine closely resemble the responses raised after infections with wild-type dengue viruses.79,80 There is growing evidence that human T cell responses directed at epitopes on non-structural proteins contribute to homotypic and heterotypic dengue virus protective immunity.81–83 Finally, live-attenuated tetravalent dengue vaccine contains genes for three of the four dengue virus NS1 proteins. A phase 3 trial testing the live-attenuated tetravalent dengue vaccine is in its third year in Brazil. The rate of accruing dengue vaccine efficacy data has
www.thelancet.com/child-adolescent Published online August 1, 2019 http://dx.doi.org/10.1016/S2352-4642(19)30205-6
For the TAK-003 efficacy trial see https://www.takeda.com/ newsroom/newsreleases/2019/ takedas-dengue-vaccinecandidate-meets-primaryendpoint-in-pivotal-phase-3efficacy-trial/
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been delayed due to an 80% reduction of dengue cases following the Zika virus epidemic of 2016–17 in Brazil.84 The outlook for live-attenuated tetravalent dengue vaccine, based on phase 2 clinical trials in humans, is that a single dose of this vaccine will raise durable, solid, protective immunity in both seronegative and seropositive individuals.
Conclusion There is reason for optimism about obtaining a successful dengue vaccine. Protection against live dengue virus challenge of vaccinated volunteers suggests that the NIH dengue vaccine soon to complete phase 3 testing will satisfy safety and efficacy requirements. Meanwhile, an intense effort is being mounted to understand the requirements for stable immune protection against homotypic and heterotypic dengue infections of humans. An important component of this research will be to understand why Dengvaxia did not provide protective immunity to seronegatives. Long lasting protection against dengue infection may require immunity to dengue NS1 protein, a component missing from Dengvaxia. An unexpected outcome of phase 3 trial of Dengvaxia might be that it provided insight into a vaccine structure that is required for tetravalent protection. Contributors Both authors contributed equally to this Viewpoint. Declaration of interests We declare no competing interests. References 1 Brady OJ, Johansson MA, Guerra CA, et al. Modelling adult Aedes aegypti and Aedes albopictus survival at different temperatures in laboratory and field settings. Parasit Vectors 2013; 6: 351. 2 Stanaway JD, Shepard DS, Undurraga EA, et al. Figure 2 in: The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis 2016; 16: 712–23. 3 Brady OJ, Gething PW, Bhatt S, et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis 2012; 6: e1760. 4 Bhatt S, Gething PW, Brady OJ, et al. The global distribution and burden of dengue. Nature 2013; 496: 504–07. 5 Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 476–81. 6 Nimmannitya S, Halstead SB, Cohen S, Margiotta MR. Dengue and chikungunya virus infection in man in Thailand, 1962-1964. I. Observations on hospitalised patients with hemorrhagic fever. Am J Trop Med Hyg 1969; 18: 954–71. 7 Bokisch VA, Top FH Jr, Russell PK, Dixon FJ, Muller-Eberhard HJ. The potential pathogenic role of complement in dengue hemorrhagic shock syndrome. N Engl J Med 1973; 289: 996–1000. 8 Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Arch Virol 2013; 158: 1445–59. 9 Puerta-Guardo H, Glasner DR, Espinosa DA, et al. Flavivirus NS1 triggers tissue-specific vascular endothelial dysfunction reflecting disease tropism. Cell Reports 2019; 26: 1598–613. e8. 10 Glasner DR, Puerta-Guardo H, Beatty PR, Harris E. The good, the bad, and the shocking: the multiple roles of dengue virus nonstructural protein 1 in protection and pathogenesis. Annu Rev Virol 2018; 5: 227–53. 11 Sierra B, Triska P, Soares P, et al. OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans. PLoS Pathog 2017; 13: e1006220.
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12 Guzman MG, Kouri G, Bravo J, Valdes L, Vazquez S, Halstead SB. Effect of age on outcome of secondary dengue 2 infections. Int J Infect Dis 2002; 6: 118–24. 13 Wilder-Smith A, Ooi E-E, Horstick O, Wills B. Dengue. Lancet 2019; 393: 350–63. 14 Gubler D. The emergence of epidemic dengue fever and dengue hemorrhagic fever in the Americas: a case of failed public health policy. Rev Panam Salud Publica 2005; 17: 221–24. 15 Wilder-Smith A. Risk of dengue in travelers: implications for dengue vaccination. Curr Infect Dis Rep 2018; 20: 50. 16 Hammon WM, Rudnick A, Sather GE. Viruses associated with epidemic hemorrhagic fevers of the Philippines and Thailand. Science 1960; 131: 1102–03. 17 Halstead SB, Nimmannitya S, Yamarat C, Russell PK. Hemorrhagic fever in Thailand; recent knowledge regarding etiology. Jpn J Med Sci Biol 1967; 20 (suppl): 96–103. 18 Halstead SB, Shotwell H, Casals J. Studies on the pathogenesis of dengue infection in monkeys. II. Clinical laboratory responses to heterologous infection. J Infect Dis 1973; 128: 15–22. 19 Halstead SB, Chow J, Marchette NJ. Immunologic enhancement of dengue virus replication. Nat New Biol 1973; 243: 24–26. 20 Halstead SB, O’Rourke EJ. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med 1977; 146: 201–17. 21 Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis 2010; 10: 712–22. 22 Ubol S, Halstead SB. How innate immune mechanisms contribute to antibody-enhanced viral infections. Clin Vaccine Immunol 2010; 17: 1829–35. 23 Boonnak K, Dambach KM, Donofrio GC, Marovich MA. Cell type specificity and host genetic polymorphisms influence antibody dependent enhancement of dengue virus infection. J Virol 2011; 85: 1671–83. 24 Kliks SC, Nimmannitya S, Nisalak A, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 1988; 38: 411–19. 25 Halstead SB. Immunological parameters of togavirus disease syndromes. In: Schlesinger RW, ed. The togaviruses, biology, structure, replication. New York: Academic Press, 1980: 107–73. 26 Libraty DH, Acosta LP, Tallo V, et al. A prospective nested casecontrol study of dengue in infants: rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model. PLoS Med 2009; 6: e1000171. 27 Chau TN, Anders KL, Lien le B, et al. Clinical and virological features of dengue in Vietnamese infants. PLoS Negl Trop Dis 2010; 4: e657. 28 Rothman AL. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nat Rev Immunol 2011; 11: 532–43. 29 Yacoub S, Wertheim H, Simmons CP, Screaton G, Wills B. Microvascular and endothelial function for risk prediction in dengue: an observational study. Lancet 2015; 385: S102. 30 Young PR, Hilditch PA, Bletchly C, Halloran W. An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients. J Clin Microbiol 2000; 38: 1053–57. 31 Schlesinger JJ, Brandriss MW, Walsh EE. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1987; 68: 853–57. 32 Libraty DH, Young PR, Pickering D, et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002; 186: 1165–68. 33 Modhiran N, Watterson D, Muller DA, et al. Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med 2015; 7: 304ra142. 34 Beatty RP, Puerta-Guardo H, Killingbeck SS, Glasner DR, Harris E. Dengue virus non-structural protein 1 triggers endothelial permeability and vascular leak that can be inhibited by anti-NS1 antibodies. Sci Transl Med 2015; 7: 304ra141.
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35 Glasner DR, Ratnasiri K, Puerta-Guardo H, Espinosa DA, Beatty PR, Harris E. Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components. PLoS Pathog 2017; 13: e1006673. 36 Puerta-Guardo H, Glasner DR, Harris E. Dengue virus NS1 disrupts the endothelial glycocalyx, leading to hyperpermeability. PLoS Pathog 2016; 12: e1005738. 37 Suwarto S, Sasmono RT, Sinto R, Ibrahim E, Suryamin M. Association of endothelial glycocalyx and tight and adherens junctions with severity of plasma leakage in dengue infection. J Infect Dis 2017; 215: 992–99. 38 Tang TH, Alonso S, Ng LF, et al. Increased serum hyaluronic acid and heparan sulfate in dengue fever: association with plasma leakage and disease severity. Sci Rep 2017; 7: 46191. 39 Gordon A, Kuan G, Mercado JC, et al. The Nicaraguan pediatric dengue cohort study: incidence of inapparent and symptomatic dengue virus infections, 2004–2010. PLoS Negl Trop Dis 2013; 7: e2462. 40 Endy TP, Chunsittiwat S, Nisalak A, et al. Epidemiology of inapparent and symptomtic acute dengue virus infection: A prospective study of primary school children in Kamphaeng Phet, Thailand. Amer J Epidemiol 2002; 156: 40–51. 41 WHO and the Special Programme for Research and Training in Tropical Diseases. Dengue guidelines for diagnosis, treatment, prevention and control: new edition. Geneva: World Health Organization, 2009. 42 Pongpanich B, Kumponpant S. Studies of dengue hemorrhagic fever. V. Hemodynamic studies of clinical shock associated with dengue hemorrhagic fever. J Pediatr 1973; 83: 1073–77. 43 Bhamarapravati N, Tuchinda P, Boonyapaknavik V. Pathology of Thailand haemorrhagic fever: a study of 100 autopsy cases. Ann Trop Med Parasitol 1967; 61: 500–10. 44 Gamble J, Bethell D, Day NP, et al. Age-related changes in microvascular permeability: a significant factor in the susceptibility of children to shock? Clin Sci (Colch) 2000; 98: 211–16. 45 Trung DT, Thao le TT, Hien TT, et al. Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg 2010; 83: 774–80. 46 Hadinegoro SR, Arredondo-Garcia JL, Capeding MR, et al. Efficacy and long-term safety of a dengue vaccine in regions of endemic disease. N Engl J Med 2015; 373: 1195–206. 47 WHO. Dengue vaccine: WHO position paper, July 2016 – recommendations. Vaccine 2017; 35: 1200–01. 48 Kalayanarooj S, Rothman AL, Srikiatkhachorn A. Case management of dengue: lessons learned. J Infect Dis 2017; 215 (suppl 2): S79–S88. 49 Wills BA, Nguyen MD, Ha TL, et al. Comparison of three fluid solutions for resuscitation in dengue shock syndrome. N Engl J Med 2005; 353: 877–89. 50 Wills BA, Oragui EE, Stephens AC, et al. Coagulation abnormalities in dengue hemorrhagic fever: serial investigations in 167 Vietnames children with dengue schock syndrome. Clin Infect Dis 2002; 35: 277–85. 51 Lye DC, Archuleta S, Syed-Omar SF, et al. Prophylactic platelet transfusion plus supportive care versus supportive care alone in adults with dengue and thrombocytopenia: a multicentre, open-label, randomised, superiority trial. Lancet 2017; 389: 1611–18. 52 Wilder-Smith A, Hombach J, Ferguson N, et al. Deliberations of the Strategic Advisory Group of Experts on Immunization on the use of CYD-TDV dengue vaccine. Lancet Infect Dis 2019; 19: e31–38. 53 WHO. Global advisory committee on vaccine safety, 15–16 June 2016. Wkly Epidemiol Rec 2016; 91: 341–18. 54 WHO. A toolkit for national dengue burden estimation. Geneva: World Health Organization, 2018. 55 WHO. Informing vaccination programs: a guide to the design and conduct of dengue serosurveys. Geneva: World Health Organization, 2017. 56 Nascimento EJM, George JK, Velasco M, et al. Development of an anti-dengue NS1 IgG ELISA to evaluate exposure to dengue virus. J Virol Methods 2018; 257: 48–57. 57 Sridhar S, Luedtke A, Langevin E, et al. Effect of dengue serostatus on dengue vaccine safety and efficacy. N Engl J Med 2018; 379: 327–40. 58 WHO. 457 Dengue vaccine: WHO position paper – September 2018. Wkly Epidemiol Rec 2018; 93: 457–76.
59 Osorio JE, Huang CY, Kinney RM, Stinchcomb DT. Development of DENVax: a chimeric dengue-2 PDK-53-based tetravalent vaccine for protection against dengue fever. Vaccine 2011; 29: 7251–60. 60 Osorio JE, Velez ID, Thomson C, et al. Safety and immunogenicity of a recombinant live attenuated tetravalent dengue vaccine (DENVax) in flavivirus-naive healthy adults in Colombia: a randomised, placebo-controlled, phase 1 study. Lancet Infect Dis 2014; 14: 830–38. 61 Rupp R, Luckasen GJ, Kirstein JL, et al. Safety and immunogenicity of different doses and schedules of a live attenuated tetravalent dengue vaccine (TDV) in healthy adults: a phase 1b randomized study. Vaccine 2015; 33: 6351–19. 62 Osorio JE, Wallace D, Stinchcomb DT. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone. Expert Rev Vaccines 2016; 15: 497–508. 63 Huang CY, Butrapet S, Pierro DJ, et al. Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 2000; 74: 3020–28. 64 Huang CY, Kinney RM, Livengood JA, et al. Genetic and phenotypic characterization of manufacturing seeds for a tetravalent dengue vaccine (DENVax). PLoS Negl Trop Dis 2013; 7: e2243. 65 Halstead SB, Marchette N. Biological properties of dengue viruses following serial passage in primary dog kidney cells: studies at the University of Hawaii. Am J Trop Med Hyg 2003; 69 (suppl 6): 5–11. 66 Bhamarapravati N, Yoksan S. Live attenuated tetravalent dengue vaccine. Vaccine 2000; 18 (suppl 2): 44–47. 67 Sanchez V, Gimenez S, Tomlinson B, et al. Innate and adaptive cellular immunity in flavivirus-naive human recipients of a live-attenuated dengue serotype 3 vaccine produced in Vero cells (VDV3). Vaccine 2006; 24: 4914–26. 68 Vaughn DW, Hoke CH Jr, Yoksan S, et al. Testing of a dengue 2 live-attenuated vaccine (strain 16681 PDK 53) in ten American volunteers. Vaccine 1996; 14: 329–36. 69 Butrapet S, Huang CY, Pierro DJ, Bhamarapravati N, Gubler DJ, Kinney RM. Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5’ noncoding region and nonstructural proteins 1 and 3. J Virol 2000; 74: 3011–19. 70 Kinney RM, Butrapet S, Chang GJ, et al. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 1997; 230: 300–08. 71 Durbin AP, Whitehead SS. Next-generation dengue vaccines: novel strategies currently under development. Viruses 2011; 3: 1800–14. 72 Whitehead SS. Development of TV003/TV005, a single dose, highly immunogenic live attenuated dengue vaccine; what makes this vaccine different from the Sanofi-Pasteur CYD vaccine? Expert Rev Vaccines 2016; 15: 509–17. 73 Durbin AP, Kirkpatrick BD, Pierce KK, et al. A single dose of any of four different live attenuated tetravalent dengue vaccines is safe and immunogenic in flavivirus-naive adults: a randomized, double-blind clinical trial. J Infect Dis 2013; 207: 957–65. 74 Kirkpatrick BD, Durbin AP, Pierce KK, et al. Robust and balanced immune responses to all 4 dengue virus serotypes following administration of a single dose of a live attenuated tetravalent dengue vaccine to healthy, flavivirus-naive adults. J Infect Dis 2015; 212: 702–10. 75 Kirkpatrick BD, Whitehead SS, Pierce KK, et al. The live attenuated dengue vaccine TV003 elicits complete protection against dengue in a human challenge model. Sci Transl Med 2016; 8: 330ra36. 76 Durbin AP, Kirkpatrick BD, Pierce KK, et al. A 12-month interval dosing study in adults indicates that a single dose of the NIAID tetravalent dengue vaccine induces a robust neutralizing antibody response. J Infect Dis 2016; 214: 832–25. 77 Smith SA, de Alwis R, Kose N, Durbin AP, Whitehead SS, de Silva AM, et al. Human monoclonal antibodies derived from memory B cells following live attenuated dengue virus vaccination or natural infection exhibit similar characteristics. J Infect Dis 2013; 207: 1898–908. 78 Tsai WY, Durbin A, Tsai JJ, Hsieh SC, Whitehead S, Wang WK. Complexity of neutralization antibodies against multiple dengue viral serotypes after heterotypic immunization and secondary infection revealed by in-depth analysis of cross-reactive antibodies. J Virol 2015; 89: 7348–62.
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79 Weiskopf D, Angelo MA, Bangs DJ, et al. The Human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes. J Virol 2015; 89: 120–28. 80 Angelo MA, Grifoni A, O’Rourke PH, et al. Human CD4+ T Cell responses to an attenuated tetravalent dengue vaccine parallel those induced by natural infection in magnitude, HLA restriction, and antigen specificity. J Virol 2017; 91: e02147–16. 81 Weiskopf D, Cerpas C, Angelo MA, et al. Human CD8+ T-cell responses against the 4 dengue virus serotypes are associated with distinct patterns of protein targets. J Infect Dis 2015; 212: 1743–51.
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82 Tian Y, Sette A, Weiskopf D. Cytotoxic CD4 T cells: differentiation, function, and application to dengue virus infection. Front Immunol 2016; 7: 531. 83 Elong Ngono A, Chen HW, Tang WW, et al. Protective role of cross-reactive CD8 T cells against dengue virus infection. EBioMedicine 2016; 13: 284–93. 84 Lopes TRR, Silva CS, Pastor AF, Silva Junior JVJ. Dengue in Brazil in 2017: what happened? Rev Inst Med Trop Sao Paulo 2018; 60: e43. © 2019 Elsevier Ltd. All rights reserved.
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Dengue infection and advances in dengue vaccines for children Scott B Halstead, Leonila F Dans
Dengue viruses are endemic in most tropical and subtropical countries where they produce disease ranging from a mild fever to a severe, potentially fatal vascular permeability syndrome. We reviewed the status of development and testing in children of three vaccines designed to protect against the four dengue viruses. The first dengue virus vaccine, Dengvaxia, now licensed in 20 endemic countries, the EU and the USA, provides protection against severe dengue in seropositive individuals but increases the risk for naive recipients to develop severe dengue and to be hospitalised. We discuss mechanisms and implications of shortcomings of the licensed vaccine and describe the structure and attributes of two other dengue virus vaccines. Based upon human dengue challenge studies, one of these vaccines promises to deliver solid, long-lasting immunity after a single dose. Because dengue virus infections are ubiquitous in residents and visitors to tropical countries, in the absence of a protective vaccine paediatricians should recognise the early signs and clinical presentation of severe dengue, understand its pathophysiology and appropriate management.
Introduction There are four closely related dengue viruses (designated DENV-1, 2, 3, and 4). All dengue viruses cause an acute febrile disease and are transmitted by the bite of Aedes aegypti, an anthropophilic mosquito that infests virtually all tropical and subtropical countries.1 Hundreds of millions of people are infected every year with outcomes that are variable and unique (figure).3,4 From the perspective of human health, the most important feature of dengue is the ability of a first infection, or passively acquired dengue antibodies, to increase the severity of the next dengue virus infection.5 This immunopathological phenomenon, termed anti body dependent enhancement (ADE), is coupled with a late-in-illness vascular permeability.6 Vascular permeability during dengue infections, identified as the dengue vascular permeability syndrome, allows fluid and small macromolecules to escape circulation and is accompanied by thrombocytopenia, altered haemostasis, elevated liver enzymes, elevated concentrations of cytokines or chemokines and activation of complement.6–8 Compelling evidence exists to suggest that much of this damage is produced by circulation during the febrile phase of a toxic viral non-structural protein 1 (NS1).9,10 For reasons not fully understood, not all humans are equally at risk to this immunopathological outcome. Attack rates are substantially lower among dengue virus-infected subSaharan Africans than among European or Asian populations.11 Moreover, the syndrome is most severe in young children, the elderly, and those with pre-existing conditions.12,13 Despite the fact that A. aegypti was largely eradicated from the Americas during the successful effort to conquer urban yellow fever in the early 20th century, sustained control of this mosquito has proved impossible.14 Dengue is a major human infectious disease in endemic countries and a substantial cause of febrile disease in tourists to tropical destinations.15 As a result there is a major effort to develop protective vaccines. However, the success of vaccines has been impacted by the dengue immuno pathology phenomenon. This Viewpoint looks at the status of vaccine development, testing, and introduction
in the context of what is known about disease causation and innate clinical responses to dengue infection.
Dengue immunopathology
Lancet Child Adolesc Health 2019 Published Online August 1, 2019 http://dx.doi.org/10.1016/ S2352-4642(19)30205-6 Uniformed Services University of the Health Sciences, Bethesda, MD, USA (Emeritus Prof S B Halstead MD); and Departments of Pediatrics and Clinical Epidemiology, College of Medicine, University of the Philippines, Manila, Philippines (Prof L F Dans MD) Correspondence to: Prof Scott B Halstead, Rockville, MD 20852, USA [email protected]
Until the 1950s, dengue was known as a disease that produced epidemics of self-limited febrile exanthemata with little mortality. This pattern changed when shock and gastrointestinal haemorrhage started being reported in association with dengue infection.16 That severe dengue might be an immunological phenomenon was suggested when it was discovered that dengue haemorrhagic fever and dengue shock syndrome accompanied a second heterotypic dengue virus infection.17 Investigating this phenomenon in rhesus monkeys, the rates of viraemia were higher during second infection than first infections with the same dengue virus.18 In addition, dengue virus grew more abundantly in cultures of peripheral blood Key messages • Three dengue vaccines for children are in or have completed phase 3 clinical testing. • Two of these vaccines are molecular chimeras, splicing structural genes of the four dengue viruses into the non-structural genes of yellow fever vaccine virus (Dengvaxia [Sanofi Pasteur]) or DENV-1, 3, and 4 genes into the DENV-2 vaccine virus (TAK-003 [Takeda Pharmaceuticals]). • The third vaccine, live-attenuated tetravalent dengue vaccine (US National Institutes of Health, NIH), contains mutagenized DENV-1, 3, and 4 and a DENV-4-and-2 chimera. A single dose solidly protected seronegative humans from recombinant non-parental DENV-2/3 challenge in phase 2b clinical trials. • In a phase 3 trial in ten dengue-endemic countries Dengvaxia provided protection against severe dengue in dengue virus seropositive children but sensitised seronegative children for risk of enhanced disease during breakthrough infections. • Dengue virus NS1 circulating at high concentrations during dengue virus infections is directly toxic to endothelial cells. • Dengvaxia contains no dengue virus NS1s while the NIH dengue vaccine contains DENV-1, 3, and 4 non-structural 1 proteins. Clinical efficacy trial results suggest that dengue virus non-structural 1 protein immunity might be important for protection. • Until dengue has been controlled paediatricians should have the knowledge to accurately diagnose acute dengue, recognise warning signs of severe dengue, and be prepared to provide life-saving resuscitative treatment.
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<10 10–99 100–199 200–499 500–999 1000–1499 1500–2999 ≥3000
West Africa
Caribbean
ATG
VCT
Barbados
Comoros
Dominica
Grenada
Maldives
Mauritius
LCA
TTO
TLS
Seychelles
Eastern Mediterranean
Marshall Isl
Kiribati
Solomon Isl
FSM
Vanuatu
Samoa
Fiji
Tonga
Malta
Persian Gulf
Singapore
Balkan peninsula
Figure: Age-standardised incidence rates of dengue (per 100 000 person-years), in 2013 ATG=Antigua and Barbuda. VCT=St Vincent and Grenadines. Isl=Island. FSM=Federated States of Micronesia. LCA=St Lucia. TTO=Trinidad and Tobago. TLS=Timor Leste. Adapted from Stanaway and colleagues, 2016.2
monocytes from dengue virus-immune humans than in non-immune humans. Furthermore, enhanced dengue virus infections could be achieved regularly when IgG dengue virus antibodies were added to dengue virus in cultures of primary human monocytes.19,20 This phenomenon extends to other acute and chronic human and animal infections in which cross-linking of IgGmicrobe immune complexes with Fcγ receptors results in increased intracellular infection contributing to enhanced disease severity, a mechanism labelled intrinsic ADE (iADE).21,22 iADE differ from extrinsic ADE (eADE), a cell surface phenomenon resulting from a more efficient attachment of immune complexes to Fc receptors than the virus itself attaching to viral receptors. eADE contributes a three-fold increase while iADE a 100-fold increase in virus production.23 Maternal dengue virus antibodies might protect newborns from dengue virus infection for several months, followed by several weeks of months of increased risk to dengue vascular permeability syndrome. This ADE event is common in the highly endemic countries of southeast Asia where mothers experience two or more lifetime dengue virus infections.20 Infant dengue vascular permeability syndrome-ADE during a first dengue virus infection, fortunately, is unique in human medicine.24–27 2
Dengue disease causation Late in the febrile period of dengue vascular permeability syndrome there might be a sudden and profound capillary leak. At first it was thought that when an increased number of dengue virus-infected target cells (monocytes, macrophages and dendritic cells) was attacked by the immune elimination response, a marked release of pro-inflammatory and anti-inflammatory factors resulted.28 These factors were thought to damage capillary integrity resulting in hypovolemic shock (also called a cytokine storm).29 The cytokine storm hypothesis has now been replaced by evidence of the direct pathogenicity of dengue virus NS1. NS1 is produced during cellular infections with each of the four dengue viruses.9 Instead of remaining cell-bound, dengue NS1 circulates in great quantities as a hexamer in blood during acute phase.30 For several decades it has been known that immunisation of laboratory animals with dengue virus NS1 protected them against lethal dengue virus challenges31 and that concentrations of NS1 in serum during an early acute phase correlated directly to the ensuing disease severity during secondary dengue virus infections.32 In 2015, a molecular analogy was observed between bacterial lipopolysaccharides and that of dengue virus NS1.33 Both bacterial lipopolysaccharides
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and NS1 interact with Toll-like receptor 4 on the surface of monocytes, macrophages, and endothelial cells to release a range of cytokines and chemokines. These same mediators are detected in blood from patients with dengue vascular permeability syndrome during the acute phase. In vitro, NS1 resulted in the disruption of the integrity of endothelial cell monolayers. The authors concluded that dengue vascular permeability syndrome might be a viral protein toxicosis. NS1-mediated cyto kine release could be inhibited by the Toll-like receptor 4 antagonist lipopolysaccharides-Rhodobacter sphaeroides, suggesting an avenue for therapeutic intervention. The same observation was confirmed in an in vivo model.34 In addition, DENV-2 NS1 inoculated intra venously at physiologically relevant concentrations in sub-lethally DENV-2-infected C57BL/6 mice produced lethal vascular permeability.35 Vaccination of mice with DENV-2 NS1 protected them against endothelial leak age and death from lethal DENV-2 challenge. Mice immunised with all four dengue virus NS1 proteins were completely protected against homologous dengue virus challenges while immunisation with DENV-1 NS1 partially protected against heterologous DENV-2 challenge. In other experiments, dengue virus NS1 was shown to directly alter the barrier function of pulmonary endothelial cell mono layers through disruption of the endothelial glycocalyx-like layer by triggering the activation of endothelial sialidases, cathepsin L, and heparanase, which are enzymes responsible for degrading sialic acid and heparan sulphate proteo glycans.36 Separately, disruption of endothelial glyco calyx components had been shown to correlate with plasma leakage during severe dengue virus infection in humans.37,38 The contribution of these dengue virus NS1-induced endothelial cell intrinsic pathways was found to be independent of inflammatory cytokines but dependent on the integrity of endothelial glycocalyx components both in vitro and in vivo.35 In conclusion, NS1 toxicosis is demonstrably an efferent mechanism of vascular permeability in mice directly related to intra cellular dengue virus production that can be modified by ADE. Based upon the kinetics of human dengue virus infections, a role for NS1 in endothelial damage, complement activation, liver damage, and haemostatic abnormalities is plausible.10
Innate host responses to dengue virus infection Infection in the absence of dengue virus antibodies
In infants and children aged 1–5 years, a first dengue virus infection is commonly inapparent.39 During midchildhood, an initial dengue virus infection usually results in a short febrile disease, the dengue fever syndrome, with potential headache, flushed face, inappetence, upper respiratory symptoms, nausea, vomiting, myalgia, leukopenia, and prostration.39 First dengue virus infections in adolescents are generally more reactogenic leading to school absenteeism in
10–20% of such individuals and some hospitalisations.40 Most non-indigenous children who acquire a first dengue virus infection while traveling or residing abroad develop this syndrome.
Infection in the presence of dengue virus antibodies Dengue virus infection of infants circulating passively acquired dengue antibodies from mothers, who have had multiple dengue virus infections, or young children (1–8 years of age) immune to a single prior dengue virus infection might initially produce mild dengue fever-like symptoms. After 2–3 days of fever, these symptoms can evolve to the signs and symptoms of dengue vascular permeability syndrome, including sudden weakness, anorexia, lethargy, mid-epigastric abdominal pain, and persistent vomiting. More alarming findings are cool extremities, scattered petechiae, thrombocytopenia (<100 000 mm³), anuria, a weak and rapid pulse, liver aspartate aminotransferase or alanine aminotransferase concentrations of 1000 (IU/L) or greater, circumoral cyanosis, an increased haematocrit with concurrent decrease in platelet count, hypovolaemia, low serum albumin, and sonographic evidence of serosal effusions. Those clinical findings that precede shock have been identified as warning signs.41 During the febrile period the signs and symptoms of dengue vascular permeability syndrome might change from day to day. Major changes in blood pressure occur as dengue vascular permeability syndrome progresses, including increased peripheral vascular resistance with decreased cardiac output and normal or low central venous pressure.42 Shock is not due to congestive heart failure but to venous pooling. With increasing cardiovascular compromise, the diastolic pressure rises toward the systolic and the pulse pressure narrows. Finally, there is decompensation and both pressures disappear abruptly. Because of increased peripheral vascular resistance, shock in dengue patients frequently manifests as a narrow pulse pressure with normal systolic values. To the unwary, dengue shock might not be recognisable. Before the modern era of proactive and careful fluid resuscitation, gastrointestinal bleeding occurred frequently in shocked children. During hypotension, blood is shunted from the splanchnic to the cerebral vascular system resulting in tissue anoxia in the gastrointestinal tract. On autopsy, shock-related gastrointestinal bleeding is by diapedesis.43 Children might be more susceptible to vascular permeability shock-related gastrointestinal bleeding than adults because of age-related differences in the integrity of vascular endothelial barriers.12,44,45 Dengue vascular permeability syndrome is unstable in the youngest patients. As children approach adolescence, the frequency of dengue vascular permeability syndrome and case fatality rate decrease. The innate susceptibility of younger vaccinated children compared with older vaccinated children (to dengue vascular permeability syndrome accompanying
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breakthrough dengue virus infections) came as a surprise to phase 3 clinical trial observers.46,47 However, the phenomenon had been described during the DENV-2 epidemic in Havana, Cuba, in 1981, where 4 years earlier 45% of the population had been infected with DENV-1.12
Disease management
For the EU license see https:// globenewswire.com/newsrelease/2018/12/19/1669374/0/ en/Dengvaxia-vaccineapproved-for-prevention-ofdengue-in-Europe.html For the US FDA license see https://www.fda.gov/newsevents/press-announcements/ first-fda-approved-vaccineprevention-dengue-diseaseendemic-regions
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Because of the high volume of travel to tropical destinations, patients with acute dengue might present to any clinical practice at any time of the year, so paediatricians should always be prepared.41,48 Success ful management of dengue vascular permeability syndrome relies on meticulous regulation of parenteral fluids and colloids during the period of increased vascular leakage, together with proactive management of major bleeding.41 The physician should remember that all fluid administered will be reabsorbed and fluid overload might result. Fluid overload is believed to contribute a substantial proportion of all deaths resulting from dengue vascular permeability syndrome.48 Because the fluid lost is approximately equivalent to plasma, isotonic crystalloid solutions are recommended, except in very young infants (<6 months of age), in whom 0·45% sodium chloride can be used. From a double-blind randomised comparison of three fluids for initial resuscitation of 383 Vietnamese children with dengue shock, it was demonstrated that Ringer’s lactate is sufficient to resuscitate children with moderately severe dengue shock syndrome.49 In patients with massive plasma leakage and those who do not respond to the maximum volume of crystalloid, hyperoncotic colloid solutions, such as 10% dextran 40 in saline, are used. Iso-oncotic colloids or albumin are not recommended. Except in patients with shock, the volume of fluid administration in patients with plasma leakage is limited to maintenance fluid requirements plus 5% deficit (5% of body weight) to account for dehydration; if patients are taking oral fluids, the recommended volume reflects the total of oral and intravenous fluids.48 The coagulopathy associated with dengue infections is well described, but unfortunately the underlying mechanisms remain unclear. Severe bleeding occurs only rarely in children (almost invariably in association with profound shock), and thrombotic complications are not seen. An increase in activated partial thrombo plastin time and a reduction in fibrinogen concentrations are fairly consistent findings, together with thrombo cytopenia, and these abnormalities tend to correlate with overall severity.50 The evidence for classical dis seminated intravascular coagulation in most cases is not convincing.50 Thrombocytopenia is almost invariable in patients with dengue, with several mechanisms thought to be involved. Early bone marrow suppression combined with increased peripheral destruction of platelets
during the febrile and early convalescent phase of the disease can lead to profound thrombocytopenia with platelet count nadirs as low as 5000 per mm³ for some cases.51 However, during the recovery period platelet numbers rise promptly as production increases in the hyper-cellular marrow. Platelet transfusions are widely administered in the mistaken belief that they will prevent severe bleeding. Many physicians treat the name of the disease (dengue haemorrhagic fever) rather than the patient. In the absence of profound bleeding (ie, bleeding sufficient to warrant consideration of blood transfusion) there is no evidence that prophylactic platelet transfusions improve outcome but offer a very definite risk both of acute and longterm complications.51
Vaccines
Dengvaxia Dengvaxia, developed by Sanofi Pasteur, is the only licensed dengue vaccine. It is live-attenuated tetravalent chimeric vaccine that incorporates the structural genes of the four dengue viruses into the genome of the yellow fever vaccine. It has been tested for vaccine efficacy and safety in placebo-controlled clinical trials enrolling approximately 35 000 children, aged 2–16 years in ten dengue-endemic countries.46 Efficacy results, published in 2015, were mixed. At year 3 after the first dose, vaccine protection of 9–16-year-old children against hospitalisation was 65·5% whereas for children aged 8 years or younger the rate was 44·6%.46 In the original phase 3 report, the serostatus at the time of vaccination was known only for a small sample. Among seropositive individuals, in the age group 2–8 years, vaccine efficacy was 70·1%, while among 9–16 year olds, efficacy was 81·9%. However, outcomes were quite different in seronegative children. In 2–8 year olds protection was 14·4% whereas for 9 years and older, it was 52·5%.46 More pointedly, among 2029 vaccinated children of 5 years or younger, 20 (0·99%) were hospitalised for dengue, a substantially higher proportion than controls, in whom the proportion was two (0·2%) per 1005, a relative risk of 4·95 (p=0·03). These data led the manufacturer to recommend the vaccine be restricted to individuals of 9 years of age and older.46 Despite the evidence that Dengvaxia was asymmetrically protective and enhancing in young children, WHO’s Scientific Advisory Group of Experts (SAGE) endorsed vaccine use for children of 9 years of age and older, followed by licensing of the product in 20 dengue-endemic countries.47,52 The EU and the US FDA issued licenses for Dengvaxia to be given to dengue-seropositive individuals resident in dengue-endemic areas. However, the serological test or tests that specifically and reliably identify dengue sero positive individuals were not identified. In 2016, SAGE recommended that Dengvaxia be given to individuals of 9 years or older residing in populations, national or subnational, with a seroprevalence of 70%
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or greater.53 To provide guidance for the deployment of Dengvaxia in high endemicity populations, WHO described case sampling and statistical methods to identify areas of high endemicity where dengue virus seroprevalence could be measured using recommended designs for sampling.54,55 To evaluate the safety of giving Dengvaxia to 9–16 year olds irrespective of sero logical status at time of vaccination, Sanofi sponsored develop ment of a serological test capable of distinguishing antibodies derived from Dengvaxia from those raised by wild-type dengue virus infections.56 This test was applied to sera from a cohort representing 10% of children taking part to a phase 3 randomized controlled trial, for all of whom blood samples had been collected during month 13 post vaccination.57 This study established that dengue seronegativity, rather than age at vaccination, placed children at a significantly increased risk of acquiring severe hospitalised dengue disease 1 year or more after vaccination compared with age and serostatus-matched controls.57 Based upon these new data, SAGE, the Global Advisory Committee on Vaccine Safety, and the WHO Dengue Vaccine Working Group reconsidered their 2016 recommendations and issued the following summary statement: “For countries considering vaccination as part of their dengue control programme, pre-vaccination screening is the recommended strategy. With this strategy, only persons with evidence of a past dengue infection would be vaccinated (based on an antibody test, or on a documented laboratory confirmed dengue infection in the past). If pre-vaccination screening is not feasible, vaccination without individual screening could be considered in areas with recent documentation of seroprevalence rates of at least 80% by age 9 years”.58
TAK-003 In January, 2018, Takeda Pharmaceuticals announced the completion of phase 3 trials for their vaccine, TAK-003.59–62 Efficacy and safety data for this vaccine have not yet been published. This vaccine consists of a live-attenuated DENV-2 strain and three chimeric viruses containing the prM and E protein genes of DENV-1, 3, and 4 expressed on the backbone of the DENV-2 genome.63,64 These chimeric dengue viruses have a long history. The parental DENV-1 and 2 from Thailand, DENV-3 from the Philippines, and DENV-4 from Indonesia incorporated into the TAK-003 vaccine were provided to the US Centers for Disease Control and Prevention Vector-Borne Diseases Branch, Fort Collins from the University of Hawaii.65 There, in 1980, DENV-1, 2, and 4 had been adapted to primary dog kidney and DENV-3 to African green monkey kidney then transferred to Mahidol University, Bangkok, where they were further passaged, tested for virulence markers and shown to be attenuated and immunogenic at different passage rates in susceptible adult volunteers.65,66 The attenuated viruses were licensed to Sanofi-Aventis, tested in thousands of volunteers of all ages but ultimately abandoned due to under-attenuation of the DENV-3 strain
serially passaged in African green monkey kidney.67 Of these four viruses, the most successful vaccine candidate was DENV-2 16881 primary dog kidney 53, which produced exceptionally high rates of seroconversions in seronegative human volunteers who had minimal dengue signs or symptoms.68 The attenuating mutations were identified and an infectious cDNA clone constructed.69,70 This DENV-2 vaccine is one component and the backbone for DENV-1, 3, and 4 in the TAK-003 vaccine. The developers hope for successful protection against all dengue virus infection or disease based on the broad neutralizing antibody responses that follow two doses of TAK-003. Clinical data from phase 3 clinical trials are awaited.
Live-attenuated tetravalent dengue vaccine For nearly 20 years, staff at the National Institute of Allergy and Infectious Diseases in Bethesda (MD, USA) and the Johns Hopkins Bloomberg School of Public Health in Baltimore (MD, USA) have been designing and testing dengue vaccine candidates. Some dengue viruses were attenuated by removing nucleotides from the non-translated region of the dengue genome (Δ30). A crucial component of this development programme was that monovalent vaccine candidates were tested for immunogenicity and attenuation in seronegative human volunteers.71 The final product, live-attenuated tetravalent dengue vaccine, contains genetically attenuated DENV-1, 3, and 4 viruses, and a fourth component, a chimera of structural genes of DENV-2 inserted into DENV-4.72–74 Having received a single dose of live-attenuated tetravalent dengue vaccine, volunteers were solidly protected from viraemia, dengue rash, or anamnestic antibody response following challenge with live-attenuated DENV-2 Δ30 (Tonga 74 strain) or DENV-3 Δ30 (Sleman 78 strain), recovered from mild dengue outbreaks and having major genetic differences from parental dengue virus (Durbin A, John Hopkins Bloomberg School of Public Health, personal communication).75 That this protection is likely to be of long duration is evidenced by the solid immune response observed to a booster dose of live-attenuated vaccine given 12 months after initial dose.76 These results are complemented by evidence that a single dose of liveattenuated tetravalent dengue vaccine raises monospecific DENV-1–4 neutralizing antibodies conformationally similar to those after human infections with wild-type dengue viruses and that correlate with protection.77,78 Moreover, the T cell responses to live-attenuated tetravalent dengue vaccine closely resemble the responses raised after infections with wild-type dengue viruses.79,80 There is growing evidence that human T cell responses directed at epitopes on non-structural proteins contribute to homotypic and heterotypic dengue virus protective immunity.81–83 Finally, live-attenuated tetravalent dengue vaccine contains genes for three of the four dengue virus NS1 proteins. A phase 3 trial testing the live-attenuated tetravalent dengue vaccine is in its third year in Brazil. The rate of accruing dengue vaccine efficacy data has
www.thelancet.com/child-adolescent Published online August 1, 2019 http://dx.doi.org/10.1016/S2352-4642(19)30205-6
For the TAK-003 efficacy trial see https://www.takeda.com/ newsroom/newsreleases/2019/ takedas-dengue-vaccinecandidate-meets-primaryendpoint-in-pivotal-phase-3efficacy-trial/
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been delayed due to an 80% reduction of dengue cases following the Zika virus epidemic of 2016–17 in Brazil.84 The outlook for live-attenuated tetravalent dengue vaccine, based on phase 2 clinical trials in humans, is that a single dose of this vaccine will raise durable, solid, protective immunity in both seronegative and seropositive individuals.
Conclusion There is reason for optimism about obtaining a successful dengue vaccine. Protection against live dengue virus challenge of vaccinated volunteers suggests that the NIH dengue vaccine soon to complete phase 3 testing will satisfy safety and efficacy requirements. Meanwhile, an intense effort is being mounted to understand the requirements for stable immune protection against homotypic and heterotypic dengue infections of humans. An important component of this research will be to understand why Dengvaxia did not provide protective immunity to seronegatives. Long lasting protection against dengue infection may require immunity to dengue NS1 protein, a component missing from Dengvaxia. An unexpected outcome of phase 3 trial of Dengvaxia might be that it provided insight into a vaccine structure that is required for tetravalent protection. Contributors Both authors contributed equally to this Viewpoint. Declaration of interests We declare no competing interests. References 1 Brady OJ, Johansson MA, Guerra CA, et al. Modelling adult Aedes aegypti and Aedes albopictus survival at different temperatures in laboratory and field settings. Parasit Vectors 2013; 6: 351. 2 Stanaway JD, Shepard DS, Undurraga EA, et al. Figure 2 in: The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis 2016; 16: 712–23. 3 Brady OJ, Gething PW, Bhatt S, et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis 2012; 6: e1760. 4 Bhatt S, Gething PW, Brady OJ, et al. The global distribution and burden of dengue. Nature 2013; 496: 504–07. 5 Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 476–81. 6 Nimmannitya S, Halstead SB, Cohen S, Margiotta MR. Dengue and chikungunya virus infection in man in Thailand, 1962-1964. I. Observations on hospitalised patients with hemorrhagic fever. Am J Trop Med Hyg 1969; 18: 954–71. 7 Bokisch VA, Top FH Jr, Russell PK, Dixon FJ, Muller-Eberhard HJ. The potential pathogenic role of complement in dengue hemorrhagic shock syndrome. N Engl J Med 1973; 289: 996–1000. 8 Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Arch Virol 2013; 158: 1445–59. 9 Puerta-Guardo H, Glasner DR, Espinosa DA, et al. Flavivirus NS1 triggers tissue-specific vascular endothelial dysfunction reflecting disease tropism. Cell Reports 2019; 26: 1598–613. e8. 10 Glasner DR, Puerta-Guardo H, Beatty PR, Harris E. The good, the bad, and the shocking: the multiple roles of dengue virus nonstructural protein 1 in protection and pathogenesis. Annu Rev Virol 2018; 5: 227–53. 11 Sierra B, Triska P, Soares P, et al. OSBPL10, RXRA and lipid metabolism confer African-ancestry protection against dengue haemorrhagic fever in admixed Cubans. PLoS Pathog 2017; 13: e1006220.
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