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Effect of immune status on dengue 2 virus replication in cultured leukocytes from infants and children.

N J Marchette, S B Halstead, T O'Rourke, R M Scott, W H Bancroft and V Vanopruks Infect. Immun. 1979, 24(1):47.

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INFECTION AND IMMUNITY, Apr. 1979, p. 47-50

Vol. 24, No. 1

0019-9567/79/04/0047/04$02.00/0

Effect of Immune Status on Dengue 2 Virus Replication in Cultured Leukocytes from Infants and Children
NYVEN J. MARCHETTE,'* SCOTT B. HALSTEAD,' THOMAS O'ROURKE,' ROBERT McNAIR SCOTT,t WILLIAM H. BANCROFT,t AND VANICH VANOPRUKSt Department of Tropical Medicine and Medical Microbiology, University of Hawaii School ofMedicine, Honolulu, Hawaii 96816,' and U.S. Component, Armed Forces Research Institute of Medical Sciences, APO San Francisco, California 96346 Received for publication 22 January 1979

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Cord blood leukocytes from neonates with maternal dengue antibody supported dengue 2 virus replication in vitro; those from neonates without maternal antibody did not. Cord bloods of infants born to dengue-immune mothers contained a potent enhancing factor which gradually decayed with age and which was absent from neonates born to nonimmune mothers. Permissiveness of cultures of washed peripheral blood leukocytes from infants with maternal antibody declined steadily with increasing age in parallel with the decay of maternal antibody, and the leukocytes were no longer permissive after 10 to 12 months. The demonstration of a dengue maternal infection-enhancing factor in human cord blood from dengue-immune mothers supports the hypothesis that severe primary dengue hemorrhagic fever with shock seen in Bangkok infants is related to maternal immune status.

In South and Southeast Asia, the dengue PBL from infants were naturally more permisshock syndrome is associated with secondary sive to dengue virus replication than PBL from dengue infection (2, 5, 6). A major exception older individuals. The present study was deoccurs in infants, predominantly ages 4 through signed to determine the relationship between 1 1 months, who develop dengue shock syndrome maternal antibody and permissiveness of infant during a primary infection (5). Halstead (2) has PBL to D2V infection. suggested that "primary" dengue shock synMATERIALS AND METHODS drome in infants might be related to the acquiLeukocyte donors. PBL were obtained from two sition of maternal dengue antibody, which at high concentration protects and at low concen- populations, one in Bangkok, Thailand, in 1974 and tration "sensitizes" the infant. Recent experi- the other in Honolulu, Hawaii, in 1975 and 1976. In blood was collected from 72 infants, ages 6 mental studies have demonstrated a mechanism Bangkok, 10 to months. Maternal blood was collected at by which disease severity could be enhanced by weeks time of delivery and tested for hemagglutination maternal antibody. Dengue virus replicates in the inhibition (HI) antibodies to dengue 1 to 4 antigens. cultures of normal adult human or simian pe- Serum from every mother possessed dengue HI antiripheral blood leukocytes (PBL) when there is body. also present a low concentration of homotypic In Honolulu, cord blood was collected from 32 newdengue antiserum (7, 8, 9). In the absence of this borns whose parents were of Oriental ancestry. Eight "enhancing" antibody, dengue virus grows mothers had been born in tropical Asia, and cord blood poorly or not at all (3, 4, 12, 13). Dengue repli- from their babies contained dengue HI and neutralizcation in this in vitro system is limited to mono- ing antibody. The remaining 24 cord bloods from infants delivered to Hawaii-born Oriental women were nuclear phagocytes (8). HI antibody. Bloods from 30 dengueIn a preliminary report (4), we described the without dengue (no dengue HI antibody) Hawaii-born chilgrowth of dengue 2 virus (D2V) in cultured PBL susceptible dren, ages 10 to 14 years, were also studied. obtained from two of three passively immune Leukocyte cultures. Leukocytes were obtained infants who were 4 to 6 months old at the time from heparinized blood (20 U/ml) or from blood clots. of study. No nonimmune infants were included, Clots were finely minced with scissors and gently and it was not possible to determine whether forced through a 60-mesh stainless steel screen (Small
t Present address: Virology Division, Walter Reed Army Institute of Research, Washington, DC 20012. t Present address: Department of Pediatrics, Phramongkhutklao Hospital, Bangkok, Thailand. 47

Parts Inc., Miami, Fla.), and the leukocytes were suspended in Hanks balanced salt solution. Plasma or serum from each sample was stored at -20'C for serological tests. Leukocytes were separated from

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erythrocytes by gravity sedimentation in 6% dextran T-250 (Pharmacia, Uppsala, Sweden) (1) and then washed three times in Hanks balanced salt solution by centrifugation at 200 x g at room temperature. The washing procedure resulted in an estimated 50,000fold dilution of autologous plasma in the final PBL preparations. Washed PBL were counted in a hemacytometer, their viability was determined by trypan blue exclusion, and they were suspended to a concentration of 1.5 x 106 live mononuclear cells per ml in RPMI 1640 medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% fetal calf serum, 2 mM glutamine, 2.0 g of sodium bicarbonate per liter, 2.4 g of N-2-hydroxyethyl piperazine N'-2-ethanesulfonic acid (HEPES) per liter, 100 U of penicillin per ml, and 100 jig of streptomycin per ml. Suspensions of PBL in 1.0-ml amounts were gassed with 5% CO2 in air and incubated at 360C in 1-dram (ca. 4-ml) screw-capped vials (Wheaton Scientific Co., Millville, N.J.). Virus. Dengue 2 (16681) virus (10), prepared in LLC-MK2 cells (11) as previously described (13), was held at -70'C in 1-dram screw-capped vials. Different vials were used for each experiment, and a portion was refrozen at -70'C for subsequent infectivity titration. Infection of leukocyte cultures. Each leukocyte culture was inoculated with 0.1 ml of a D2V suspension containing 3.0 x 10' plaque-forming units. Replicate vials also received 0.1 ml of RPMI, 0.1 ml of a 1:5 dilution of autologous plasma, or 0.1 ml of a 1:5 dilution of H-187 rhesus monkey anti-D4V plasma (final dilution, 1:50). This concentration of anti-D4V plasma regularly enhanced D2V replication in nonimmune adult human PBL. Cultures were incubated at 360C, and on days 3 to 5 of incubation duplicate vials were frozen at -70'C until assayed for virus content. Titration of enhancing antibody. D2V at a multiplicity of infection of 0.1 and cord blood plasmas at final dilutions of 1:25,000 to 1:106 were added to cultures of PBL from a nonimmune adult human donor. Each experiment contained a negative control, in which virus was added to PBL cultures containing no dengue antiserum, and a positive control containing virus and a standard concentration of anti-D4V serum. All cultures were incubated at 36C, and on days 3 to 5 replicate vials were frozen at -70C until assayed for virus content. Virus assay. Thawed PBL suspensions were diluted 1:10 in phosphate-buffered saline (pH 7.9) containing 20% heat-inactivated agamma calf serum, and D2V titers were determined by a standard LLC-MK2 plaque assay (10). The virus inoculum was titered similarly. Analysis of results. Plaque counts were normalized by logarithmic transformation and analyzed by Student's group comparison or paired t test.

to 10-month-old Bangkok infants were less permissive, with mean virus titers less than 1.0 logo plaque-forming units per ml after 3 to 5 days of incubation. PBL from 30 dengue nonimmune children older than 12 months did not support D2V replication in vitro. Cultures of washed cord blood leukocytes (CBL) from each of eight neonates with maternal dengue antibody supported D2V replication to greater than 2.0 logo plaque-forming units per ml. The addition of autologous plasma at 1:50 final dilution did not further enhance D2V replication, whereas the addition of monkey antiD4V (1:50 final dilution) had a synergistic effect (Table 1). In contrast, significant D2V replication did not occur in any CBL cultures from 24 Honolulu Oriental infants born to mothers without dengue antibody either in the presence or absence of autologous plasma (Table 1). However, CBLcultures from nonimmune donors readily supported D2V growth in the presence of monkey anti-D4V at a 1:50 final dilution. Dengue 2 infection-enhancing titers were measured in seven dengue antibody-containing cord blood plasmas. A single plasma enhanced D2V infection at a dilution of 1:500,000, whereas six had enhancement titers of equal to or greater than 1:106. Cord blood plasma without dengue antibody did not enhance D2V infection at a dilution of 1:10, the lowest dilution tested.
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FIG. 1. Replication of D2V in cultures of washed

RESULTS peripheral blood leukocytes from infants 0 to 12 The growth of D2V in leukocyte cultures by months of age born to mothers with dengue antibody from children greater than 1 year of age who age group of donor is summarized in Fig. 1. and were without dengue antibody. The numbers in parCultures of washed leukocytes from each of 52 enthesis are the number of individuals tested in each newborn to 4-month-old, dengue-immune Bang- age group. The differences between age groups are kok infants supported D2V replication at levels highly significant (P < 0.01). The less-than-2-monthsbetween 2.0 and 3.0 logio plaque-forming units old group includes CBL from eight neonates born to per ml of culture. Washed PBL cultures from 5- mothers with dengue antibody.

VOL. 24, 1979

D2V REPLICATION IN INFANT LEUKOCYTES

49

TABLE 1. Mean D2V titers in cord blood leukocytes from infants born of mothers with or without dengue antibody Log1o mean virus titer (days 3
to 5) in CBL cultures supplemelted with: AutoloRPMI gous ankey plasma anti-D4V 8 4.35(C) 3.22(B) 2.90(A) Dengue immune 24 2.61 (F) 0.85(D) 0.73(E) Nonimmune a Leukocytes were cultured in complete RPMI medium containing additional RPMI, autologous plasma, or monkey anti-D4V serum. Statistical analysis by paired t test was as follows: A versus B, P > 0.10; B versus C, P = 0.10; A versus C, P < 0.02; D versus E, P > 0.10; E versus F, P < 0.02; A versus D, P < 0.01; B versus E, P < 0.01; C versus F, P < 0.01; B versus F, P = 0.40; A versus F, P = 0.50.

Maternal immu- No. studied nity status

DISCUSSION The present study provides evidence that PBL from dengue-susceptible human newborn infants spontaneously supported little or no D2V replication in vitro. In the presence of dengue antiserum, however, CBL cultures supported immune complex-initiated D2V infection of the same order of magnitude as do PBL from older nonimmune human beings (7). Thus, circulating human leukocytes, irrespective of the age of the donor, are capable of supporting D2V replication in the presence of non-neutralizing antibody. In this study no attempt was made to identify the type of blood leukocyte supporting dengue replication, but in other studies with leukocytes from adult donors infected under conditions identical to those described in these experiments mononuclear phagocytes were the only cells supporting virus growth (8). Washed CBL from neonates born to dengueimmune mothers supported D2V replication at virus titers similar to those observed in PBL from children and adults with actively acquired dengue immunity (4). Five to ten months after birth, PBL cultures from Bangkok infants (born to dengue-immune mothers) had lost most or all of their permissiveness to D2V infection. The simplest explanation of these two observations is that the more permissive cultures contained residual maternal antibody. The washing procedure resulted in approximately 50,000-fold plasma dilution, but dengue-immune cord blood plasmas enhanced infection at dilutions as high as 1:106. Thus the permissiveness of CBL to D2V replication might be the result of cord blood enhancing antibody carried into leukocyte cultures. Furthermore, the titer of passively acquired antibody is maximal at birth and progressively declines with increasing age of the infant, resulting in a concomitant age-dependent

decrease in permissiveness in the passively immunized infant. Plasma of infants with maternal dengue antibody had much higher enhancing antibody titers (1:500,000 or higher) than that customarily found in plasma of individuals with active dengue immunity (1:50,000 or lower) (7). This suggests that in cord blood plasma there may be other factors in addition to antibody that affect permissiveness to D2V infection. The enhancing factor in adult plasma is known to be immunoglobulin G (7), but that in cord blood is yet to be characterized. When anti-D4V serum was added to washed dengue-immune CBL, marked synergism in viral replication was observed (Table 1, A versus C). Since we do not usually observe an increase in enhancement of dengue infection over a broad range of dilutions above the neutralization end point, this observation implies that there are two populations of infant leukocytes which are permissive to D2V infection. One population contains cells with specific virus receptors, perhaps strongly bound cytophilic antibody which is not removed by washing. It is conceivable that this population of permissive cells is composed of or includes sensitized cells transferred from the mother to the infant, but there is no evidence for it. A second population includes cells without cytophilic antibody or other virus receptors but capable of being infected by virus-antibody complexes. The spontaneous permissiveness of washed CBL from dengue-immune neonates is reminiscent of a similar phenomenon for individuals with actively acquired immunity (4, 13). The identity of the cell-associated enhancing factor is under active study. When autologous plasma was added to the system at a final dilution of 1:50, virus replication was not enhanced to the same level as with antiD4V (Table 1, B versus C). This is most likely due to D2V neutralization by maternal dengue antibody. If dilutions of cord blood antibody above 1:50 had been used, enhanced D2V infection should have occurred. In conventional plaque reduction neutralization tests, each cord blood plasma had a D2V neutralization titer of 1:100 to 1:500 and high-titered enhancement activity well above these dilutions. These data provide further support for the hypothesis that enhanced infection of leukocytes mediated by passively transferred dengue antibody is the basis for severe primary dengue infections observed in infants in the Bangkok hyperendemic area. In this study, six cord bloods with dengue HI antibody had enhancing titers of approximately 1:106. If the half-life of maternal antibody is estimated at 21 days and this amount of enhancing antibody is present at

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beings. Proc. Soc. Exp. Biol. Med. 151:136-139. 5. Halstead, S. B., S. Nimmannitya, and S. Cohen. 1970. Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J. Biol. Med. 42:311-328. 6. Halstead, S. B., S. Nimmanitya, C. Yamarat, and P. K. Russell. 1967. Hemorrhagic fever in Thailand. Newer knowledge regarding etiology. Jpn. J. Med. Sci. Biol. 20:96-103. 7. Halstead, S. B., and E. O'Rourke. 1977. Dengue virus and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146:201217. 8. Halstead, S. B., and E. O'Rourke. 1977. Dengue virus and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. J. Exp. Med. 146:218-229. 9. Halstead, S. B., and E. O'Rourke. 1977. Antibody-enhanced dengue virus infection in primate leukocytes. Nature (London) 265:739-741. 10. Halstead, S. B., S. Udomsakdi, P. Simasthien, P. Singharaj, P. Sukhavachana, and A. Nisalak. 1970. Observations related to pathogenesis of dengue hemorrhagic fever. I. Experience with classification of dengue viruses. Yale J. Biol. Med. 42:261-275. 11. Hull, R. N., W. R. Cherry, and 0. J. Tritch. 1962. Growth characteristics of monkey kidney cell strains LLC-MK1, LLC-MK2, and LLC-MK2 (NCTC-3196) and their utility in virus research. J. Exp. Med. 115: 903-917. 12. Marchette, N. J., and S. B. Halstead. 1974. Immunopathogenesis of dengue infection in rhesus monkeys. Transplant. Proc. 6:197-201. 13. Marchette, N. J., S. B. Halstead, and J. S. Chow. 1976. Replication of dengue viruses in cultures of peripheral blood leukocytes from dengue-immune rhesus monkeys. J. Infect. Dis. 133:274-282.

birth, there would still be residual enhancing activity through month 10 or 11 of life. The natural decay rate of passively acquired enhancing antibody in infants born to dengue-immune mothers was not precisely measured in this study, but could easily be determined in a group of passively immune infants followed through the first year of life.
ACKNOWLEDGMENTS This study was supported in part by Public Health Service grant 5 R01 HD08693 from the National Institutes of Health and a grant from the Southeast Asia Regional Office, World Health Organization. We are grateful for the generous cooperation of Ralph Hale and the obstetrical nurses at Kapiolani Hospital for providing cord bloods and to Patty Iwamoto, May Tom, Susan Cate, and Carrie Uyehara for excellent technical assistance. Arwind Diwan, Department of Tropical Medicine and Medical Microbiology, University of Hawaii, supplied blood samples from Oriental children born in Hawaii.
LITERATURE CITED 1. Alexander, R. F., and A. L. Spriggs. 1960. The differential diagnosis of tumour cells in circulating blood. J. Clin. Pathol. 13:414-424. 2. Halstead, S.B. 1970. Observations related to pathogenesis of dengue hemorrhagic fever. VI. Hypotheses and discussion. Yale J. Biol. Med. 42:350-362. 3. Halstead, S. B., J. S. Chow, and N. J. Marchette. 1973. Immunological enhancement of dengue virus replication. Nature (London) New Biol. 243:24-26. 4. Halstead, S. B., N. J. Marchette, C. J. Sung, and S. Lolekha. 1976. Dengue virus replication enhancement in peripheral blood leukocytes from immune human

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