Immunology Today, Vol. ,1, No. 2, 1983

The immunopathogenesis of dengue haemorrhagic fever T. Pang and K. S. K. Lam Over the past 20 years, dengue haemorrhagic .fever ( D H F ) has been the subject of intensive epidemiological, clinical, virological and immunological investigations. Considerable debate and controversy have surrounded its causation and the probable role of immunological mechanisms in its pathogenesis. The exact cause of D H F is still uncertain and this article reviews current thinking about the problem. Although classical dengue fever has been recognized for over 200 years, the severe form of the disease dengue haemorrhagic fever (DHF) has only been recognized as such since it was discovered in the Philippines in 1953-541.2 . The disease can now be found all over South East Asia and the South Pacific and, since 1956, has resulted in 350,000 patients being hospitalized and nearly 12,000 deaths 3. D H F continues to be a major public health problem in the region and in several countries where it is not recognized currently, but where dengue viruses are actively transmitted. It remains a potential threat to approximately 1500 million people worldwide.

Dengue viruses D H F is a group B arbovirus infection transmitted by the Aedes mosquito, of which Aedes aegypti is the most important vector. The virus is classified as a flavivirus in the family Togaviridae. Four dengue serotypes (1-4) have been identified all with marked antigenic cross-reactivity 4. Cross-reactive antibody is present from the onset of secondary infection, and an anamnestic response occurs very rapidly resulting in very high titres of IgG antibody.

Dengue haemorrhagic fever D H F is found mainly in Asian children between 2 and 13 years of age. The start of the disease is marked by the abrupt onset of fever accompanied by denguelike symptoms including haemorrhagic manifestations, mostly found as petechial skin haemorrhages, hepatomegaly and circulatory disturbances 3,5. Following what appears to be the normal course for classical dengue fever, the patients' condition suddenly deteriorates; they may become hypotensive and die within a few h o u r ¢ ,5. Severe cases may also go into a condition that is referred to as dengue shock syndrome (DSS) 3.

Concepts of pathogenesis Several lines of argument have been put forward to explain the underlying pathogenesis of D H F / D S S : the 'sequential infection' hypothesis envisaging a key role for the host immune response in producing D H F / D S S ; the suggestion of alterations in the virulence potential of the virus; and the proposal that D H F / D S S is attributable to defects in immunoDepartment of Medical Microbiology, Faculty of I~ledicine, University of Malaya, Kuala Lumpur, Malaysia.

regulatory mechanisms in a host with a pre-existing parasite infection.

Sequential infection hypothesis The studies of Halstead et a/. 6.7, a s well as other investigators ~.'~, produced data consistent with the concept that a primary infection with any of the dengue viruses produced a mild, benign illness, whereas further infection with a different dengue type could result in severe, often fatal, D H F / D S S . This is the central tenet of the sequential infection hypothesis proposed by Halstead"': viz, that a person sensitized by a first dengue infection may elicit, through immunopathologic processes, the severe haemorrhagic and shock response when, months or years later, he is infected by a second type of dengue. The sequential infection hypothesis was put forward largely on the basis of very comprehensive studies carried out by Halstead and his colleagues in Bangkok, Thailand from 1958-1964 ('.11-15. The main evidence advanced in support of the hypothesis is seroepidemiological. It indicates that where D H F / D S S occurs there are two or more dengue serotypes simultaneously endemic and that D H F / D S S is nearly always a s s o c i a t e d with a s e c o n d a r y - a n t i b o d y response, i.e. the syndrome usually occurs as a result of dengue infection in a patient with pre-existing antibody. Serological evidence suggests that children with D H F / D S S exhibit a secondary immune response except for infants under one year of age who give a primary response, presumably because they have been passively immunized by maternal antibodies. It was also observed that dengue-2 virus was isolated significantly more frequently from D H F / D S S cases than from other serotypes. In direct relation to the sequential infection hypothesis several proposals have been made as to the actual immunopathologic mechanisms involved in producing the shock syndrome. As suggested by Russell ".~y, the IgG formed during the secondary response forms antigen-antibody complexes with viral antigen in the blood. The formation of such complexes then leads to massive complement activation resulting in increased vascular permeability, thus initiating the shock syndrome ",~7. In support of this contention it was found that various complement components were markedly depressed in D H F / D S S patients. One such component is C34,16 whose levels fall just before the onset o f shock and at the time antibody levels are rising tT. The levels of C3 pro-activator, C4 and C5, © E l s e v i e r B i o m e d i c a l Press 1983


Immunology Today, Vol. 4, No. 2, 1983

have also been found to be greatly reduced 4. Furthermore, the level of circulating immune complexes detected in the sera of D H F / D S S patients appears to be related to the clinical grading and severity of the disease 1~-2('. In the study of Ruangjirachuporn el al. TM, for e x a m p l e , c i r c u l a t i n g i m m u n e c o m p l e x e s , apparently dengue-specific, were detected in 80% of D H F / D S S sera. These complexes were first detected 2 days after the onset of fever, reached peak levels on days 4-5, or when the patients developed shock, and then declined 2°. Immune complexes in D H F / D S S were also found to be localized in tissue and blood 2~.22. However, despite the evidence for the presence of immune complexes, the exact nature of the antigen(s) and antibody(ies) reacting in these complexes remain unknown. In contrast to the idea of immune complexes per se initiating the shock syndrome by activation of complement, more recent investigations have suggested a role for 'enhancing' antibodies. Several key experimental observations are relevant. The most significant was the demonstration that dengue 2~-2s, and other flaviviruses 2',27, showed enhanced replication in h u m a n or simian peripheral blood leucocytes 23-2s and in macrophage cell lines 2~'.27in the presence of subneutralizing concentrations of specific antibody. A similar phenomenon has also been noted for alphaviruses and bunyaviruses 27. Although the exact mechanism of antibody mediated enhancement is still unclear, the most likely cell involved is thought to be the monocyte 3 and it is probable that Fc receptors on these susceptible cells act to promote the entry of infectious immune complexes formed in the presence of subneutralizing concentrations of antibody 2v. In-vi~o evidence for this was obtained when monkeys, previously treated with normal or dengue-immune sera, were inoculated with dengue-2 virus. Viraemia titres were found to be 3-51-fold higher in the animals which had received immune sera 2s. On the basis of these observations, the 'immunological enhancement' hypothesis has been put forward 3.2~. The hypothesis proposes that in the presence of 'enhancing' antibody, virus is engulfed and then replicates within monocytes. Owing to the mobility of the monocytes, the infection may then spread to bone marrow, liver, spleen, gut lymphoid tissues and histiocytes in the skin. It is then postulated that an 'immune elimination' response directed against dengue-infected monocytes, probably .involving killer T lymphocytes, is responsible for activating such monocytes into releasing various factors including C3b, thromboplastin and an unidentified vascular permeability factor ~,2~. The release of such factors then leads to the pathological changes seen in D H F / D S S . Although initially questioned', later investigations indicated that virusspecified antigens are incorporated into the plasma membrane of dengue-infected cells ~v thus making these cells a suitable target for a cytotoxic T-cell response. Antibody-mediated cytolysis of dengueinfected cells has been demonstrated by Russell and Brandt r but a specific cytotoxic T-cell response, as has been conclusively shown with other viruses 2'J, has

47 not been reported. From the above discussion it is clear that the true nature of the immunopathologic mechanism(s) and the identity of the pharmacologically active mediators involved remain unknown. It is quite possible also that several different mechanisms are involved which are initiated by the immune complexes depending upon the relative concentrations of antigen and antibody at a particular point of time. These may include complement activation a n d / o r replication in monocytes as envisaged by the immunological enhancement hypothesis. Alternative explanations, such as the role of cell-mediated immunity, for example, remains a relatively unexplored possibility. Additionally, the involvement of other gri~ups of mediators such as the recently identified leukotrienes 3°, must also be considered. These mediators have been shown to induce systemic hypotension, cause haemodynamic and permeability changes and affect the cutaneous microcirculation ~°. It is quite possible, for example, that the 'unidentified' vascular permeability factor referred to by Halstead -~,2~is in fact a leukotriene. Alterations in virus Despite the popularity and the apparent acceptance of the sequential infection hypothesis in many quarters 33, several objections have been raised to its validity. This issue has been the subject of considerable controversy and debate and several observations have been quoted as being incompatible with the sequential infection hypothesis. First, outbreaks of D H F / D S S with severe haemorrhagic mafiifestations and shock, which were apparently caused by primary dengue infection, have been reported in the South Pacific 31.32. Serological surveys indicated that each outbreak was caused exclusively by dengue-2 virus and furthermore, that there was no evidence of infection with any other dengue serotypes over the preceding 25 years 33. Secondly, the majority of cases of mild dengue fever in South East Asia show a secondary antibody response as high titred as in severe cases 34. Thirdly, D H F / D S S can occur in persons older than 1 year of age undergoing their first dengue infection 33. This implies that sensitization by previous dengue infection is not essential to the pathogenesis of D H F / D S S and that other factors may be involved. O n the basis of the above and other considerations, Rosen 33 has stated his objection to the sequential infection hypothesis and has proposed an alternative theory based on changes in the virus. His theory proposes that all observations on D H F / D S S can be explained by the concept that different strains of dengue viruses of all four serotypes vary in their pathogenic potential and that D H F / D S S are relatively rate consequences of dengue infection 33. As has been suggested by H a m m o n 3~, such changes to produce variant, 'haemorrhagic' strains of dengue viruses could be achieved by mutation and genetic instability perhaps operating together with selective forces of both their vertebrate and invertebrate hosts. Alternatively, opportunity for recombination phenotypic mixing resulting in a variant virus may arise

Immunology Today, Vol. 4, No. 2, 1983

48 when a double infection occurs with two different dengue viruses 3s. It is also possible for two different viruses, one dengue, one non-dengue, to act synergistically to produce the more serious disease. In relation to the development of such variant viruses, Rudnick 34,3~has made the interesting observation that in areas where both A. aegypti and A. albopictus transmit dengue, severe cases of disease were correlated with the distribution of A. aegypti. By contrast only mild dengue cases were detected in areas where A. albopictus is present. This has led to the suggestion that the mild, endemic dengue strains normally transmitted by the native vector, A. albopictus, may become modified into a more virulent variant when transmitted by the introduced vector, A. aegypti, i.e. some interaction may be necessary between the virus and the two vectors to produce the suggested modification 34. Despite the proposals discussedabove relating to virus modification, the experimental evidence in support of the concept is relatively meagre. Although many studies have been carried out to characterize the antigenic structure of dengue viruses 4.r.3('.37, many questions remain unanswered. For example, detailed antigenic characterization of dengue viruses isolated from D H F / D S S cases as compared with those isolated from mild, classical dengue fever has not been carried out. In relation to Rudnick's proposal mentioned previously, the possibility of mosquito vectors bringing about some antigenic modification of the virus should also be investigated. More importantly, the nature of the antigen or antigenic determinants and the immunoglobulin subtypes reacting in immune complexes and those involved in immunological enhancement of dengue replication have not yet been investigated. Furthermore, it would be of interest to determine the number of 'enhancing' determinants shared between sequential dengue infections. To a large extent, attempts to study these questions have been hampered by several problems including the lack of a good experimental model, the absence of useful markers for virulence and the marked cross-reactivity of antisera preparations to the four dengue serotypes. With respect to the last problem, the use of monoclonal antibodies prepared by the hybridoma technique ~-4° could make batteries of determinantspecific antibody preparations available. These antibodies would be of great value in mapping the antigenic determinants in dengue viruses and in identifying those determinants involved in the shock syndrome. It would also be relevant to the development of vaccines.

Defects in immunoregulation In a recent article 4~, Pavri and Prasad put forward a hypothesis which attempts to link D H F / D S S with pre-existing parasite infection. They propose that the dual infection with dengue virus and parasite results in a depletion of T-suppressor cells resulting in augmented IgG and IgE production which leads to a combination of type III and type 1 hypersensitivity reactions, vasoactive-mediator release and the shock

syndrome 4~. Although some indirect evidence has been quoted in support of this theory, i.e., elevated IgE levels in the sera of D H F / D S S patients 42, there seems to be no direct evidence pertaining to the numbers and function of T-suppressor cells and the status of parasite infection in D H F / D S S cases. Furthermore, experimental studies in mice seem to indicate that T-suppressor cells remain at normal levels during dengue infection 43,q4.

Unanswered questions In addition to the points already mentioned, it should be pointed out that several other questions remain in relation to the D H F / D S S problem which any working hypothesis must attempt to explain: (1) the age-specific incidence of D H F / D S S ; (2) the absence of D H F / D S S in regions known to have had epidemics caused by different dengue serotypes; are there racial, ethnic or economic factors involved?; (3) the apparent failure of other group B arboviruses to 'sensitize' an individual; (4) the more frequent association of dengue-2 virus with cases of D H F / D S S . At the present time there are no, or only partial, answers to these questions.

Conclusions and synthesis In conclusion, it is evident that a great deal of emphasis has been placed on studying the involvement of immunological mechanisms in the pathogenesis of D H F / D S S . The evidence strongly supports a role for immunopathological processes in the terminal shock syndrome. However, the epidemiology and aetiology of the earlier severe haemorrhagic disease, short of the terminal shock syndrome, remain unclear. As has been suggested by H a m m o n 3~, D H F / D S S appears unlikely to be simply a continuum of the normal spectrum of classical dengue fever. It seems rather to be due to the introduction of new factor(s) to the host-parasite relationship. In view of the available evidence it seems logical, at this point in time, to continue to consider the possibility of changes in the causal agent as being one of these new factors; it seems a little unreasonable to place all emphasis on the immunologic disease hypothesis as the explanation for the total disease spectrum. In all, it is rather appealing to view the proposals of Halstead, Russell and coworkers on the one hand and those of Rosen, H a m m o n and Rudnick on the other, not as two opposing theories but rather as probable explanations for processe s at different ends of the disease spectrum. An alteration in the pathogenic potential of the virus could then be regarded as prerequisite for triggering off the unique set of reactions, primarily immunological in nature, which results in the shock syndrome of D H F / D S S . Within this scheme, cases of primary D H F / D S S mentioned previously could conceivably be due to a variant form of dengue in which the minor • haemorrhagic lesions (petechiae) are exacerbated and extended in severity. The late appearance of the lesions would also suggest that petechiae may be a minimal manifestation of the same immunopathologic processes proposed in the sequential infection

Immunology Today, Vol. g, .No. 2, 1983

hypothesis ~. In addition to variant forms of dengue virus, other factors, such as defective immune response or alteration of antigen-antibody ratios at a critical stage of the disease, may also be involved. Finally, it seems appropriate to ask the question why the problem of causation of DHF/DSS is of such importance. Firstly, although the disease can be controlled by controlling the main vector (A. aegypli), this is no longer a simple task. Resistance to insecticides, cost consid.erations and logistic problems all contribute to the difficulties of vector control. Secondly, the sequential infection hypothesis proposed by Halstead and colleagues has had very important implications in relation to the possible use of vaccines to control the disease. If DHF/DSS is due entirely to immunologic sensitization then immunization would obviously be the worst possible method of controlling the disease. Finally, and as borne out by the preceding discussion, many fundamental questions remain to be answered with regard to definition of risk factors for DHF/DSS, mechanisms of pathogenesis of the disease and the role of virus strain and host immune response in production of the shock syndrome. It is because of our lack of knowledge as to what constitutes the 'exact antecedents of DHF/DSS, and thus the potential threat of disease to millions of people around the world, that intensive research into the problem seems justified. This research will hopefully provide insights into the fundamental aetiology and pathogenesis of DHF/DSS and in turn lead to a more rational approach to therapy and the development of an effective vaccine for safe and lasting prophylaxis in man.

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The immunopathogenesis of dengue haemorrhagic fever.

Over the past 20 years, dengue haemorrhagic fever (DHF) has been the subject of intensive epidemiological, clinical, virological and immunological inv...
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