We took advantage of the exceptional epidemiologic situation in Cuba, where the population initially ... history of a primary infection by dengue virus 1 or 2.
Original Report Long-term to dengue
memory cellular immune response virus after a natural primary infection
Beatriz Sierra,(‘) Gissel Garcia,(l) Ana B. Perez,(‘) Luis Morier,(l) Mayling Alvarez,(‘) and Maria G. GuzmBn(i)
Rayner
Rodriguez,(l)
Objectives: This study was conducted to examine the memory T-cell response to dengue virus 20 years after a primary infection. We took advantage of the exceptional epidemiologic situation in Cuba, where the population initially suffered two large successive epidemics due to dengue virus 1 and 2 respectively over a 4-year period. Thereafter, no dengue virus circulation was subsequently observed, except for the Santiago de Cuba municipality. Design: T-cell response was evaluated in peripheral blood mononuclear cells (PBMCs) from 20 individuals with history of a primary infection by dengue virus 1 or 2. Methods previously shown to induce lymphoproliferation of CD4’ memory T-cell subpopulations were used. We evaluated the proliferative responses generated in those PBMCs after stimulation with dengue virus 1,2,3 and 4 antigens in a serotype-specific and serotype-crossreactive way. Results: Serotype-specific and serotype-crossreactive lymphoproliferative responses in all PBMCs donated by dengue immune donors were observed. The serotype-crossreactive response for dengue 2 was stronger than for the rest of the serotypes. Conclusions: This is the first report of cellular memory lymphocyte years after a primary infection by dengue. Int J Infect
Dis 2002;
6: 125-128
INTRODUCTION
Although the circulation of dengue virus has been widely documented in the region of the Americas, reports of clinical syndromes caused by dengue virus infection in Cuba have been sporadic.’ However, in 1977 Cuba suffered an extensive epidemic of dengue fever (DF), caused by dengue virus type 1.2 This epidemic was followed by a dengue hemorrhagic feveridengue shock syndrome (DHF/DSS) outbreak in 1981, caused by dengue 2 virus, which produced a high number of cases and fatalities.3 The circulation of dengue had not been subsequently observed until January 1997, when a focal dengue epidemic in Cuba produced 205 classical DHF/DSS cases, with 12 deaths in persons 18 years and older in Santiago de Cuba, the vast majority of them with a secondary-type antibody response. 3,4The epidemic was almost controlled by August 1997. Effective vector control measures arrested the spread of the disease to other parts of the country.” For these reasons, the Cuban experience may be considered unique: two large successive epidemics due to
(‘)Department DiseasesTropical
of Virology, PAHO/WHO Medicine Institute,
Collaborating Havana, Cuba.
Center
for Viral
Address correspondence to Dr. Beatriz de la C. Sierra, Laboratory of Immunology, Department of Virology, Institute for Tropical Medicine ‘Pedro Kouri’, Autopista Novia de1 Mediodia, Km 6, PO Box 601, Marianao 13, Havana, Cuba. E-mail: siebet2002@yahoo,es. Corresponding
Editorial
response specific for dengue virus detected 20
Office:
New
York
dengue virus types 1 and 2 respectively occurred 4 years apart, followed by several years in which no dengue virus circulation was detected. These exceptional circumstances offered us the possibility to explore the memory T-cell immune response to dengue virus 20 years after the primary infection. MATERIALS
AND METHODS
We studied 20 Cuban individuals from Havana City, with previous infection by dengue 1 and dengue 2 during the epidemics which occurred in Cuba in 1977 and 1981, respectively, with titers of l/40 or higher of antibodies to dengue virus by ELISA.6 Ten of them were primary cases of dengue 1 and 10 of dengue 2, as shown using plaque reduction neutralization: sera were tested by neutralization test at dilutions between l/10 and l/1000 for their ability to neutralize dengue virus by semimicro methods in BHK21 cells.’ PBMCs from five individuals without antibodies to dengue virus were used as negative controls. Dengue 1 (Hawaii strain), dengue 2 (New GuineaC strain), dengue 3 (H87 strain) and dengue 4 (H242 strain) were used to produce antigens for the lymphoproliferation assay (LPA). These antigens were purified from infected suckling-mouse brains by equilibrium centrifugation in a 1560% (w/u) sucrose gradient in TNE.s The negative control antigen was prepared similarly but from uninfected suckling-mouse brains. Purified antigens were inactivated, sterilized, aliquoted, and frozen at -70°C.
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All PBMCs were purified by density gradient centrifugation.9 To measure the proliferative response, cells (1 x lo5 in 100 uL of supplemented RPM1 1640) were cultured for 5 days at 37°C in the presence of dengue virus 1,2,3 and 4 or control uninfected suckling-mouse brain (20 ug/mL) and phytohemagglutinin (5 ug/mL) in supplemented RPM1 1640 medium and pulsed with 1 uCi of tritiated thymidine ([3H]TdR, Amersham, UK) for 6 h before harvest. [3H]TdR incorporation was measured in a liquid scintillation counter and stimulation index was calculated.lO Anti-CD8 and Anti-CD4 antibodies directly conjugated to fluorescein isothiocyanate (Becton Dickinson Co., Mountain View, CA, USA) were used to count CD8+ and CD4+ PBMCs in a fluorescence-activated cell sorter (FACS 440; Becton Dickinson Co.).”
lation determined by FACS showed that 57.54% of the cells were CD3+CD4+ T-cells, and 21.79% were CD3+ CD8’ (mean values). DISCUSSION The generation of a cellular immune response after a primary dengue virus infection was reported by Kurane et al in 1989.‘* Serotype-specific and serotype-crossreactive memory T-lymphocytes have been detected in patients after recovering from natural dengue virus infection or following immunization with live-attenuated virus vaccines.13 These previous studies were performed in areas with high dengue prevalence, where frequent reinfections are likely to occur. However, in the reviewed literature, no studies exploring the long-term memory cellular immune response to dengue virus after a natural primary infection have been found. The population in Cuba suffered two large epidemics due to dengue 1 (1977) and dengue 2 (1981) and no other outbreaks were recorded after this time, with the exception of a small outbreak in the Santiago de Cuba municipality.4 Immediately after this second epidemic, a passive dengue surveillance system was established.The data recorded from 1982 to 1996 demonstrate that no dengue viruses had circulated in the country for those 14 years, since no anti-flavivirus IgG antibodies were detected in sera from children born after 1982.i4
RESULTS PBMCs obtained from each donor with dengue primary infection showed adequate proliferative responses to PHA and to dengue virus antigens (Figures 1 and 2). Serotype-specific stimulation in all cases and crossreactivity in most of them were detected. Control antigen did not induce proliferative responses. PBMCs from negative controls showed no proliferation in response to any of the dengue virus antigens. PBMC phenotypes after dengue virus antigen stimu-
Dl immune
donors
M immune
donors
$ fr % T6 .k >
Controls
Figure 1. Average pt$tohemagglutinin
of stimulation (PHA).
index
of the
individuals
0
50
Average
of stimulation
immune
to dengue
100 index 1 (Dl)
and
dengue
2 (D2)
and
controls
in response
to
and controls
in response
to
DZ immune donors Dl immune donors
Controls
Figure 2. Average the four serotypes
of stimulation index of dengue virus.
I
I I
0
2
of the
individuals
4
immune
I
I
6
8
to dengue
1 (Dl)
I
10
and
Stimulation index dengue
2 (D2)
Long-term memory cellular immune response to dengue virus after a natural primary infection I Sierra et al
The above-mentioned situation offered us the possibility to explore the long-term memory T-cell response to dengue virus after a natural primary infection. Our results confirm the existence of a memory T-cell response that exhibits serotype-crossreactive proliferative response to dengue virus 20 years after the primary dengue infection. To our knowledge, this is the first time that a longstanding T-cell response to dengue virus has been reported. Kurane et alI1 have previously analyzed the dengue virus-specific human T-lymphocyte clones for dengue serotype-crossreactive specificities.They found a stronger crossreactive response to dengue 3 in dengue l-immune individuals. In our study, however, we noted the strongest serotype-crossreactive response to dengue 2 in dengue l-immune donors. The analysis of the dengue complex interactions by comparison of the protein amino acid sequences showed that dengue 1, dengue 3 and dengue 4 are antigenically more closely related to each other than to dengue 2.i5 However, taking into account the important role of sequential infection as a risk factor for the development of the DHF, secondary heterotypic infection by dengue 2 has frequently been associated with DHF in dengue limmune patients.16 In the Cuban experience, in both DHF epidemics, 1981 and 1997, the sequence of infection was the same: dengue 1 followed by dengue 2.3,4 It seems that this observation reflects an immunogenic relationship between dengue 1 and dengue 2 serotypes. On the other hand, it is a recognized fact that the severity was greater in both the 1981 and 1997 Cuban epidemics when compared with those that occurred in the American region, caused by the same dengue virus serotypes and strain. l’,i* Our results regarding the cellular immune response could be related to that event, Probably, the same viral and host factors do not operate in the same manner among genetically different populations. Kurane and EnnisI and Rothman and Ennis20 have hypothesized that the dengue virus-specific, serotypecrossreactive T-lymphocytes, which are activated during secondary dengue virus infection, play an important role in the pathogenesis of DHF. They are stimulated to produce cytokines like IFN-?I, which has a pivotal role in this model for DHF immunopathogenesis, and also permit, by lysing dengue virus-infected monocytes, the release of high levels of cytokines and chemical mediators that induce malfunction of vascular endothelial cells, which in turn produces plasma leakage and shock.2’ Owing to the intensive dengue infection surveillance carried out from 1981 to 1997, it became clear that DHF/DSS cases in the dengue 2 epidemic in Santiago de Cuba in 1997 occurred in persons infected initially with dengue 1 in 1977-79. 3,4 This epidemic showed that a primary infection is a risk for the development of the severest clinical presentation, even 20 years after the primary infection.
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Taking into account the above-mentioned model of immunopathogenesis, our results may be the first confirmation of the existence of memory crossreactive Tlymphocytes 20 years after infection. This could be partially responsible for the 205 classical DHF/DSS cases with 12 deaths in persons 18 years and older observed in Santiago de Cuba, 20 years after the primary infection. More interestingly, it has been statistically demonstrated that a secondary dengue 2 infection occurring at an interval of 20 years after a dengue 1 primary infection can be more severe than a secondary dengue 2 infection occurring 4 years after a dengue 1 primary infection.” The case fatality rate was nearly five-fold higher in 1997 than in 1981. A hypothesis is that hostrelated factors, such as time-related changes in immune response, may be responsible for the severity differences observed.22 The heterotypic long-duration memory Tcell response, confirmed in this work, could be involved in this time-enhanced severity. ACKNOWLEDGMENTS
The authors thank Dr Alan Rothman, Center for Infectious Disease and Vaccine Research, University of Massachusetts, for offering helpful advice on the realization of this work. The authors also thank Dr Virginia Cape, Department of Pathology, Institute for Tropical Medicine ‘Pedro Kouri’, Havana, Cuba for critical reviewing and valuable advice on the preparation of this paper. REFERENCES 1. Gubler DJ. Dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G, eds. Dengue and dengue hemorrhagic fever. New York: CAB International Press,1997. 2. M&s, I? Dengue fever in Cuba in 1977: some laboratory aspects.In: Dengue in the Caribbean. Scientific Publication No. 375. Washington DC: Pan American Health Organization, 1979:40-43. 3. Kouri GP, Guzman MG, Bravo JR, Triana C. Dengue hemorrhagic fever/dengue shock syndrome: lessons from the Cuban epidemic, 1981. Bull WHO 1989; 67(4):375-380. 4. Kouri G, Guzman MG, ValdCs L, et al. Re-emergence of dengue in Cuba: the 1997 epidemic in Santiago de Cuba, 1997. Emerg Infect Dis 1998; 4:89-92. 5. ValdCs L, Guzman MG, Kouri G, Delgado J, Carbonell I y otros. Epidemiologia de1 dengue y fiebre hemorragica de1 dengue en Santiago de Cuba, 1997. Pan Am J Public Health 1999; 6:16-25. 6. Vazquez S, Bravo J, Perez AB, Guzman MG. ELISA de inhibition. Su utilidad para clasificar un case de dengue. Rev Cub Med Trop 1997; 49(2):10&112. 7. MD, Halstead SB, Repic PM, Putvatana R, Raybourne N. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells. Comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microb 1985; 22(2): 250-254.
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8. Sierra BC, Rodriguez R, Garcia G, Perez AB, Guzman MG. Evaluation of two different procedures to obtain dengue virus antigen for cellular immunity assays.Clin Diagn Lab Immunol (submitted). 9. Boyam A. Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Lab Invest 1968; 21 (suppl):77-89. 10. Knight SC. Lymphocyte proliferation assays. In: Klaus GGG, ed. Lymphocytes: a practical approach. IRL Press, 1987:189-207. 11. Kurane I, Meager A, Ennis FA. Dengue virus specific human T cell clones: serotype cross-reactive proliferation, interferon gamma production and cytotoxic activity. J Exp Med 1989; 170:763-775. 12. Kurane I, Innis B, Nisalak A, et al. Human T cell response to dengue virus antigens. Proliferative responses and interferon gamma production. J Clin Invest 1989; 83506-513. 13. Dharakul T, Kurane I, Bhamarapravati N, et al. Dengue virus specific memory T cell responses in human volunteers receiving a live attenuated dengue virus type 2 candidate vaccine. J Infect Dis 1994; 170(1):27-33. 14. Guzman MG, Kouri G, Bravo J, Soler M, Vazquez S, Morier L. Dengue hemorrhagic fever in Cuba,1981: a retrospective seroepidemiologic study. Am Trop Med Hyg 1990; 42(2):179-184.
15. Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 1990; 179: 479-493. 16. Halstead SB. Antibody, macrophages, dengue virus infection, shock and haemorrhage: a pathogenetic cascade. Rev Infect Dis 1989; 11(4):5830-5839. 17. Guzman MG, Kouri G, Valdes L, Bravo J, Vazquez S, Halstead SB. Enhanced severity of secondary dengue 2 infection occurring at an interval of 20 compared with 4 years after dengue 1 infection. Int J Epidemiol (in press). 18. Pan American Health Organization. Dengue and dengue hemorrhagic fever in the Americas: an overview of the problem. Epidemiol Bull 1992; 13(1):9-10. 19. Kurane I, Ennis FA. Immunopathogenesis of dengue virus infection. In: Gubler DJ, Kuno G, eds. Dengue and dengue hemorrhagic fever. New York: CAB International Press, 1997. 20. Rothman AL, Ennis F. Immunopathogenesis of dengue hemorrhagic fever. Virology 1999; 257:1-6. 21. Kurane I, Ennis FA. Cytokines in dengue virus infections: role of cytokines in the pathogenesis of dengue hemorrhagic fever. Semin Virol 1994; 5:443448. 22. Guzman MG, Kouri G, Vazquez S, Rosario D, Bravo JR, Valdez L. DHF epidemics in Cuba, 1981 and 1997: some interesting observations. Dengue Bull WHO 1999,23:3943.
