1. DENGUE: EPIDEMIOLOGY PART II
SCOTT B HALSTEAD, MD
Part II of the Dengue Epidemiology lecture describes the intrinsic host factors that contribute to control the severity of the clinical response accompanying primary and secondary dengue infections and describes other epidemiological observations that document the contribution of antibody dependent enhancement to control the outcome of dengue infections. .
Director, Research
PEDIATRIC DENGUE VACCINE INITIATIVE

2. EPIDEMIOLOGY Risk factors for severe disease.
Sequential dengue infection (includes antigenic structure of virus)
Race
Age
Host genetic factors
Nutritional status
Sex
Host factors that affect the severity of dengue disease include race, age, host genetic factors and nutritional status. Sequential infections have been presented in DENGUE EPIDEMIOLOGY Part I.

3. Race Caucasian & Asian vs African. At least 5:11,2
Guzman MG et al. AJTMH 42: , 1990.
Halstead SB et al AJTMH 65:180, 2001
Perhaps the strongest intrinsic control of dengue severity among humas has been attributed to a dengue resistance gene in individuals of subsaharan African origin. This phenomenon was described during the 1981 DHF/DSS outbreak in Cuba and verified by the absence of DHF/DSS in Haiti, a dengue-hyperendemic country with a predominantly black population.

4. BLACKS ARE RELATIVELY RESISTANT TO SEVERE DENGUE ILLNESS
A human resistance gene seems to explain the observation that while all dengue virus types circulate in Africa no DHF/DSS cases or outbreaks of DF have been reported.
While all four dengue virus types circulate in Africa few outbreaks of dengue fever and none of DHF/DSS have been reported from Africa south of the Sahara.

5. RACE - SANTIAGO DE CUBA OUTBREAK, 1997
Note that percent of DHF cases or deaths in whites, mixed or Asians exceed the distribution of these ethnic groups in the open Cuban population, while black are under-represented in both categories.

6. Haiti, showing the location of Port-au-Prince, capital city and site where UN troops landed in From 517 suspected dengue cases, 185 viruses were isolated: DENV 1 = 65; DENV 1 = 73; DENV 4 = 47.

7. DENGUE NEUTRALIZING ANTIBODIES BY AGE IN 210 CHILDREN RESIDENT
IN PORT AU PRINCE, HAITI, 1996.
High levels of multitypic dengue neutralizing antibodies circulate in Haitian children, ages 6 – 13 years. Monotypic dengue neutralizing antibodies to each of the four dengue viruses were found. Dengue virus types 1,2 & 4 had been recovered from UN personnel in including DHF/DSS-associated Asian genotype DENV 2 (Halstead et al 65: , 2001).
N =(46) (40) (36) (41) (27) (13) (10) (4)

8. AGE

9. DHF - EFFECT OF AGE, 1981 Cuba Outbreak
Fortuitously, the only dengue viruses ever to infect the Cuban population ages 3 – 40 years, were DENV 1 in and DENV 2 in Epidemiological studies showed that rates of infection for these viruses were the same in all age and ethnicity categories (Guzman MG et al AJTMH 42: , 1990) and thus, age-specific DHF/DSS attack rates per 10,000 secondary dengue infections measure intrinsic age-specific susceptibility to dengue vascular permeability. Note that case fatality rates in the elderly exceed reported DHF/DSS attack rates.
GUZMAN MG et al. Int J Infect Dis 6:18, 2002

10. CAPILLARY FRAGILITY
As measured in Vietnamese volunteers, intriinsic capillary permeability varies with age. Young infants exhibit the most fragile capillaries. Capillary walls strengthen in young adults but become more fragile with advancing age.
Gamble J et al. Biochem Soc Med Res Soc 98:211-6, 2000.

11. GENETIC ASSOCIATIONS Susceptibility Resistance
HLA1: HLA-A* HLA-A*0203
HLA-B* HLA-B*52
HLA A24 HLA A33
Vit D2: t allele/352
FcRγII3:
DCSIGN4: CD 209 promoter
TNFα5: TNF 308
1. Loke H et al. JID 184: , 2001
2. Stephens HA et al. Tissue Antigens 60: , 2002.
3. Loke H et al. AJTMH 67:102-6, 2001
4. Sakuntabthai A et al. Nat Genetics 37:507-13, 2005
5. Fernandez-Mestre MT et al. Tissue Ag 64:468-72, 2004
A variety of genetic markers studied in individuals with secondary dengue infections have been statistically significantly associated with mild (resistance) or severe (susceptibility) disease.

12. NUTRITIONAL STATUS

13. NUTRITIONAL STATUS OF DHF CASES vs. CONTROLS
Note the marked reduction in prevalence of 1st, 2nd and 3rd degree malnutrition in children with DHF/DSS compared with children from the same population with other infectious diseases or healty controls.

14. Effect of nutritional status on dengue disease severity1
Well nourished children:
highly susceptible to severe disease
Malnourished: protected against severe disease (protein-calorie malnutrition grade 2 and 3)
1. Thisyakorn U et al. CID 16: , 1993
Remarkably, grade 2 or 3 protein-calorie malnutrition protects against severe dengue vasculopathy.

15. SEX

16. SEX RATIOS BY DENGUE SYNDROME Bangkok Children’s Hospital, 1962-64
Bangkok females 4 years and older are at increased risk to DSS. Previous studies had established that dengue virus infection rates were equal in male and females in the open population of Bangkok children (Halstead SB et al AJTMH 18: , 1969).

17. DHF/DSS during primary dengue infections.
Perhaps the most remarkable demonstration of ADE in vivo is exhibited by infants who acquire classical DHF/DSS during primary dengue infections that occur during the second half of the first year of life.

18. DHF/DSS in infants, identical to but more severe than DHF/DSS in children
Higher case fatality rates, resuscitation requires more fluid per Kg body weight than in older children with 2o infection.
Hung NT et al AJTMH 72:370, 2005
Circulating cytokines and cytokine levels during acute phase similar to those in older children during 2o infection.
Hung NT et al JID 189:221, 2004
DHF/DSS in infants is difficult to diagnose and often under-recognized in smaller community hospitals. In Thailand, Myanmar (Burma), Vietnam and Indonesia, primary infections in infants contributes no less than 5% of total DHF/DSS cases. Passive dengue antibodies from mothers have been identified as risk factor. But, as cases occur predominantly during a six month period, attack rates are higher in this age group than in any other.

19. DSS in a 6 month-old infants with hepatomegaly. Vietnam
As in older children, DHF/DSS occurs in well nourished infants.
DSS in a 6 month-old infants with hepatomegaly. Vietnam

20. INFANT DHF/DSS
Age distribution of infants hospitalized at the Queen Sirikit National Institute of Child Health (Bangkok Childrens Hospital). From Halstead et al. EID 212: , 2002.

21. The age distribution of DHF/DSS in infants explained as a function of dengue antibodies that protect at birth, but as they wane they enhance dengue infections and finally disappear; at months, a period of very low DHF/DSS attack rates, infants for the first time are susceptible to unmodified primary dengue infections. (Halstead SB et al. Yale J Biol Med 42: ,1970; Halstead SB Amer J Epidemiol 114: ,1981; Kliks S et al AJTMH 38: , 1988)

22. WHY DO MATERNAL ANTIBODIES ENHANCE DENGUE DISEASE?
CENTRAL ROLE OF MACROPHAGES IN SUPPORTING DENGUE INFECTIONS IN HUMANS
DHF/DSS in infants comprises the strongest evidence that IgG1 dengue antibodies enhance the severity of dengue disease. Enhanced viremias are produced in monkeys previously given polyclonal or monoclonal dengue antibodies at non-neutralizing concentrations (Halstead SB JID 140: , 1979; Goncalvez et al PNAS 104:9422-7, 3007)

23. Dengue viruses are adapted to grow in dendritic cells,
monocytes and macrophages.
Cells of mononuclear phagocyte lineage are the predominant site of dengue infection, in vivo. Non-neutralizing antibodies help to target dengue viruses to these cells. All human viral hemorrhagic fever viruses target monocytes/macrophages but because of strong tropism do not require assistance from heterotypic antibodies.
Complexed with antibodies dengue viruses enter FcR-bearing cells with great efficiency.

24. IMMUNE ENHANCEMENT OF DENGUE INFECTION (Antibody-Dependent Enhancement)
In the presence of dengue ADE antibody:
increased rate of infection
increase in the number of infected cells.
increased production of viruses per cell.
ADE has been studied extensively, in vitro (Halstead SB et al. J Exp Med 146:218-29, 1977; Chareonsirisuthigul et al. J Gen Virol 88:365-77, 2007).

25. ADE IN HUMAN DENGUE: DEN virus load and disease severity
Children with fevers of 1-2 days duration were enrolled in study, bled on five consecutive days and viremic blood titered in mosquitoes. Peak viremia titers were observed 2-3 days prior to onset of shock or vascular permeability.

26. DEN 3 VIREMIA ●--● DSS ▲- ▲DHF □--□ DF
Virus titers in acute phase blood samples were assayed by quantitative PCR. Viremias on day 2 were 10-fold higher in children who subsequently developed DSS than in those with DF.
LIBRATY DH et al JID 185:1213, 2002

27. DISEASE SEVERITY CORRELATES WITH CELLULAR INFECTION
Peak viremia is a surrogate for the number of dengue-infected cells. Viremia levels diminish once antibodies are released into circulation. During secondary dengue infections, anamnestic antibody production begins early. Secondary viremias are rapidly reduced and are shorter in duration that primary viremias (Vaughn et al JID ).

28. Schematic distribution of dengue 2 viruses
in blood and tissues of 31 rhesus monkeys.
Tissue infection by dengue 2 viruses peaks just after viremia ends. In humans vascular permeability occurs at about the time that viremia ends (defervescence) and cellular infection is maximal based upon the monkey model (Marchette et al JID 128:23-30, 1973; Halstead SB in Schlesinger WR Togaviruses Academic Press NY pp ).

29. Vascular permeability in dengue infections is an efferent phenomenon secondary to T cell attack on dengue virus-infected cells. This activates complement and generates a cytokine “perfect storm” that correlate with disease severity. An increased dengue-infected cell mass correlates with T cell response and cytokine production (Pang T et al. Immunol Cell Biol 85: 43-5, 2007)

30. In endemic areas, DHF/DSS annual outbreaks differ in severity and size.

31. MYANMAR: VARYING CFR YEAR CASES DEATHS CFR 1986 2,192 111 5.06 1987
7,292
222
3.04
1988
1,181 65
5.5
1989
1,196 52
5.78
1990
6,318
182
2.8
1991
8,055
305
3.7
1992
1,514 40
2.63
Significant differences in case fatality rates do not correlate with the number of hospitalizations. Possible explanations include differences in case fatality rates associated with different dengue infection sequences. There were not measured during this period.

32. DENGUE VIRUSES, BANGKOK 1973 - 2001
The most upper figure shows dengue confirmed hospitalized cases by 2months in 1973 –2001. There has been general upward trend and more cases some year than others. Each year, peak hospitalization rates occur during the month of July thru October. The lower 4 graphs show isolations by dengue serotype for serotype 1, 2, 3 and 4 by 2 months. You will see clearer in the next slide.

33. WHY?
Possible effect of
ADE
Heterotypic immunity
Replacement of serotypes
Clade extinctions
These possibilities have been explored in mathematical models, most are based upon the hospital epidemiological data from Bangkok or all of Thailand.

34. EFFECT OF ADE ON EPIDEMIC CYCLES
“Enhancement of infection may generate a complex and persistent cyclical or chaotic epidemic behavior ….and coexistence of mutiple strains”
Ferguson N et al. The effect of antibody-dependent enhancement on the transmission dynamics and persistence of mutiple-strain pathogens.Proc Natl Acad Sci USA 96:790-4, 1999

35. EFFECT OF HETEROTYPIC IMMUNITY ON EPIDEMIC CYCLES
8-10 year epidemic cycles are accompanied by clade extinctions.
Mathematical model suggests that heterotypic immunity is responsible.
Adams B et al PNAS 103: , 2006

36. SEROTYPE REPLACEMENT
DENV -1 replaced DENV 2, 3, 4. Related to stochastic event due to low transmission in ?
Thu HM et al. Myanmar denge outbreak associated with displacement of serotypes 2, 3 and 4 by dengue 1. Emerg Infect Dis 10:693-7, 2004.

37. CLADE EXTINCTIONS DUE TO STOCHASTIC EVENTS
In Myanmar, clades B and C of genotype I DENV -1 circulated with clade A genotype III during the 1990s. After 1998, clade A disappeared leaving only clades B and C.
Thu HM et al Lineage extinction and replacement in dengue type 1 virus populations are due to stochastic events rather than to natural selection. Virol 336:163-72, 2005.
In Thailand, clades of DENV -3 circulating prior to 1992 disappeared and were replaced by two lineages with common ancestor. Earlier extinctions, 1963, 1973?
Wittke V et al. Extinction and rapid replacement of strains of dengue 3 virus during an interepidemic period. Virol 301: , 2002.