Immunology and Cell Biology (2007) 85, 4345

& 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00
www.nature.com/icb

REVIEW

Of cascades and perfect storms: the

immunopathogenesis of dengue haemorrhagic
fever-dengue shock syndrome (DHF/DSS)
Tikki Pang1, Mary Jane Cardosa2 and Maria G Guzman3

The past four decades has witnessed a consolidation of the original observations made in the 1970s that dengue haemorrhagic
fever (DHF) and dengue shock syndrome (DSS) have an immunological basis. Following reinfection with a dengue virus of
different serotype, severe disease is linked to high levels of antibody-enhanced viral replication early in illness which is followed
by a cascade of memory T-cell activation and a storm of inflammatory cytokines and other chemical mediators. These
compounds are released mainly from T cells, monocytes/macrophages and endothelial cells, and ultimately cause an increase
in vascular permeability. The consolidation of the evidence has been largely due to several important prospective sero-
epidemiological studies in areas endemic for DHF/DSS, which have shown that risk of severe disease is significantly higher in
secondary dengue infections. These advances have underscored the fact that DHF/DSS pathogenesis is a complex, multifactorial
process involving cocirculation of various dengue virus serotypes and the interplay of host and viral factors that influence disease
severity. The continued search to define risk factors in susceptible populations must be combined with the new techniques of
molecular virology and innovative approaches in vaccine design to achieve the ultimate objective of developing a safe and
effective vaccine.
Immunology and Cell Biology (2007) 85, 4345. doi:10.1038/sj.icb.7100008; published online 28 November 2006

Keywords: dengue haemorrhagic fever; immunopathogenesis; cytokines

The dramatic pathological potential of cytokines was recently demon-
strated during a phase I clinical trial of TGN1412 in the UK.1,2
Injection of small doses of this immunomodulatory drug, a huma-
nized monoclonal antibody acting as a superagonist to the CD28
receptor of T cells, resulted in a severe adverse reaction and hospita-
lization of six volunteers, at least four of whom suffered major organ
failure, severe depletion of regulatory T cells and are likely to suffer
immune system-related problems for life. Although full details have
yet to emerge, it seems likely that such a catastrophic reaction was
due to the triggering of a cytokine storm following massive T-cell
activation.
Such a phenomenon, however, would not be new, unexpected nor
surprising to those familiar with the disease syndromes caused by the
dengue viruses, especially in its most severe manifestation dengue
haemorrhagic fever and dengue shock syndrome (DHF/DSS), which is
characterized by high fever, increased vascular permeability, bleeding,
liver enlargement, circulatory failure and accompanied by thrombocy-
topenia and haemoconcentration. Members of the flaviviridae, the four
serotypes of dengue are a major public health threat in the developing
world. Acknowledged as the most important human arboviral infec-
tion in the world, it is responsible for 50100 million cases of febrile
illness annually, including more than 500 000 cases of DHF/DSS with
a case fatality rate of around 20%.3 Dengue is now thought to be
endemic in more than 100 countries in different parts of the tropical
developing world and more than two billion people are at risk of
acquiring dengue infection.4 The economic impact of dengue illness
is thought to be considerable, especially in the developing world.5
The possible involvement of T cells in the pathogenesis of DHF/DSS
was suggested more than two decades ago6 but much has happened
since then to refine our understanding of the key events and
mechanisms involved, including the central role played by enhancing
antibodies and inflammatory cytokines. In the past four decades since
the initial studies in Thailand,7 a convincing and irrefutable body
of evidence has been assembled which points to the fact that a large
majority of DHF/DSS cases is closely linked to the immune system,
specifically individuals experiencing secondary infections with dengue
viruses.8
Although much has been learnt in recent years about dengue virus
structure and biology, viral receptors and interactions with host cells,
the intrinsic virulence of some dengue virus strains, the effect of viral

1Department of Research Policy and Cooperation, World Health Organization, Geneva, Switzerland; 2Institute of Health and Community Medicine, Universiti Malaysia Sarawak,

Kuching, Malaysia and 3Department of Virology, PAHO/WHO Collaborating Center for the Study of Dengue and its Vectors, Instituto de Medicina Tropical Pedro Kouri, Havana,
Cuba
Correspondence: Dr T Pang, Department of Research Policy and Cooperation, World Health Organization, Ave Appia 20, Geneva 1211, Switzerland.
E-mail: pangt@who.int
Received 19 September 2006; accepted 26 September 2006; published online 28 November 2006
 Immunopathogenesis of DHF/DSS
T Pang et al
44
infection on innate immunity, and the role of other viral and host
factors (e.g. genetic factors, nutritional status, ethnicity, underlying
chronic disease, sequence of virus infections, role of dendritic cells),
this brief review focuses solely on the main immunological events
which underpins the serious manifestation of disease (DHF/DSS)
following infection with the dengue viruses in humans. References
cited are illustrative and selective rather than comprehensive, and the
reader is referred to more detailed reviews which have been written
on the subject.813
ANTIBODY-DEPENDENT ENHANCEMENT AND OTHER
ANTIBODY-MEDIATED EVENTS
During secondary infections with a heterotypic dengue virus serotype,
extensive evidence from both in vitro and in vivo studies14 strongly
suggest that pre-existing, subneutralizing, and non-protective levels of
dengue antibodies enhance virus replication in Fc-receptor bearing
cells, especially monocytes and macrophages. IgG antibodies have
been implicated15 as well as enhancement via IgM and complement
C3 receptors.16 Enhanced replication of dengue viruses in these target
cells are probably responsible for the high levels of viraemia at the
early stage of illness which have been correlated with DHF inci-
dence.17,18 Prospective sero-epidemiological and population-based
studies carried out during the DHF epidemics in Cuba in 1981,
1997 and 20012002,11 and in other endemic areas,19 confirmed
that secondary dengue infection was a significant risk factor in more
than 97% of severe cases.11 In addition, enhancing antibodies could
affect disease severity as long as 20 years after the primary infection,
especially during dengue-2 and dengue-3 infection following a pri-
mary infection with dengue-1 virus.20 However, all dengue serotypes
have been shown to be capable of causing severe dengue illness.21
In contrast, milder disease is associated with lower viral loads and
high levels of pre-existing heterotypic neutralizing antibodies during
secondary infection.
In addition to antibody-dependent enhancement (ADE), cross-
reactive antibodies directed against the dengue nonstructural NS-1
protein have also been implicated in causing damage to endothelial
cells by inducing them to undergo nitric oxide-mediated apoptosis.22
In vitro studies indicate that cytokine and chemokine production in
endothelial cells, including interleukin-6 (IL-6), IL-8 and MCP-1
(monocytes chemoattractant protein 1), increased after treatment
with anti-NS1 antibodies23 suggesting the possibility of an autoim-
mune pathogenesis in DHF/DSS based on molecular mimicry.24 Also,
antibodies potentially crossreactive to plasminogen (due to a similarity
to dengue envelope glycoprotein and a family of clotting factors) could
play a role in the aetiology of haemorrhage in DHF/DSS.25 Antibodies
to NS1 persist long after dengue infection has resolved, yet do not
cause pathology. It may thus be important to understand how such
autoimmune mechanisms are limited to the time when an active
infection is occurring. More recent, albeit preliminary, research has
also hinted at the possible role of antibody-dependent cell-mediated
cytotoxicity (ADCC).26 ADCC activity was detected in acute sera from
DHF patients but not from patients with the milder dengue fever, thus
hinting at a possible role for ADCC in DHF/DSS pathogenesis. More
research is needed to fully understand the full significance and role
of ADCC in DHF/DSS pathogenesis and its relative importance with
respect to T-cell activation.
T-CELL ACTIVATION
There is strong evidence of T-cell activation in vivo during dengue virus
infection, and such activation of CD4+ and CD8+ T cells is greater in
patients with DHF than those with the milder dengue fever.810
Immunology and Cell Biology
Following the ADE of viral replication in monocytes and macro-
phages, viral antigens are presented in conjunction with human
lymphocyte antigen molecules on the surfaces of such cells. This is
then followed by activation of CD4+ and CD8+ memory T cells,
sensitized during a previous infection, leading to proliferation and
release of proinflammatory cytokines such as interferon gamma
(IFNg) and tumour necrosis factor alpha (TNFa).27 These cytokines
can act directly upon vascular endothelial cells resulting in plasma
leakage. Studies of the extent of T-cell activation in Thai children with
DHF/DSS indicated that severe disease is associated with high levels
of T-cell activation accompanied by massive apoptosis.28
There has also been considerable interest in the specificity of these T
cells, and detailed study of T-cell responses in dengue-infected Thai
children concluded that they show a low affinity for the infecting virus
and a higher affinity for another virus serotype, presumably from
a previous infection, thus mimicking the phenomenon of original
antigenic sin.28 Crossreactive dengue-specific T cells were recently
shown to induce high levels of cytokine production, which may lead
to increased vascular permeability.29
CYTOKINE CASCADES
Although a complete and detailed picture is yet to emerge, it is
currently believed that, following massive activation of memory T
cells, a cytokine cascade targeting vascular endothelial cells is primarily
responsible for the critical pathologic event of creating an endothelial
sieve effect leading to fluid and protein leakage. Higher concentra-
tions of such cytokines, released mainly by T cells, monocytes/
macrophages and endothelial cells8 have indeed been shown to exist
in the serum of patients with DHF/DSS and, in addition to IFNg and
TNFa, IL-2, IL-6, IL1-b and IL-8 are also elevated. These findings have
been supported by more recent studies which analysed sera from
DHF/DSS patients in Vietnam,30 India31 and Cuba32 which showed
the presence of elevated levels of IFNg, TNFa and IL-10.
It is also well known that cytokines can induce the release and
production of other cytokines this complex interactive network of
induction results in further increases in the levels of cytokines and
other chemical mediators.8 Cytokines also often have synergistic effects,
for example, TNFa, IFNg and IL-1 together induce a larger increase in
permeability compared to when each cytokine is acting alone.33 In
addition, IFNg is also able to upregulate the expression of Fcg receptors
on monocytes and macrophages, thus further facilitating viral replica-
tion. The cytokine response is clearly closely linked to T-cell activation
and it is likely that the generally accepted ADE-viral replication-T-cell
activation-cytokine release sequence is not a strictly linear process but
rather a complex interplay of self-reinforcing pathological events which
ultimately manifests in DHF/DSS, increased vascular permeability and
circulatory failure. However, this complex interaction does not explain
DHF/DSS during primary infection observed in infants less than one
year of age born from dengue-immune mothers.
The likelihood that cytokines, rather than virus, are responsible for
damage to endothelial cells during DHF was suggested by an immuno-
pathological study of human tissues from patients with DHF34 where
the presence of dengue antigens in endothelial cells was thought to be
due to deposition of virus-antibody complexes rather than the viral
replication which occurs in monocytes and macrophages.
COMPLEMENT AND OTHER MEDIATORS
Complement activation has also been proposed as a key underlying
event in the immunopathogenesis of DHF/DSS but its cause has
remained unknown. Recent studies, however, suggest that large
amounts of dengue NS1, complement anaphylatoxin C5a, and the

terminal complement complex SC5b-9 were present in pleural fluids
of patients with DHF/DSS and may contribute to the vascular leakage
which occurs in these patients.35
In addition to complement, other mediators such as histamine,
tissue plasminogen activator (tPA) and, more recently, macrophage
migration inhibitory factor (MIF) have also been implicated. Of
particular importance may be the release of tPA which has been
demonstrated following dengue virus infection of endothelial cells.36
In the case of MIF, a recent report showed that higher serum levels
were detected in all DHF/DSS patients who died than in those who
survived or had the milder dengue fever, leading the authors to
propose that MIF could be a potential predictor of disease severity
and clinical outcome of disease.37
IMPLICATIONS FOR PREVENTION AND CONTROL
Despite laudable efforts to control and prevent dengue disease through
vector control measures, improved disease management, better public
education and awareness, and public health measures, it is acknowl-
edged that a safe and effective vaccine is still the most effective means
of achieving long-term, sustainable control. However, a safe and
effective vaccine against a disease with such strong immunological
underpinnings clearly poses considerable challenges. Among other
requirements, a safe vaccine needs to elicit long-lasting protection
against all four dengue serotypes as incomplete protection against any
one serotype may enhance the risk of acquiring DHF/DSS among
vaccinated individuals. Bearing in mind differences in the intrinsic
biology and replication rate of various dengue serotypes, this is a
daunting challenge. Another major challenge relates to the continued
lack of clarity about correlates of protection and the absence of a good
experimental model for DHF/DSS. Although new approaches such as
humanized mouse models are being actively pursued,38 and show
symptoms such as fever, rash and thrombocytopenia, they suffer from
the fact that they do not mimic the DHF/DSS symptoms as seen in
humans. More details on the issues facing vaccine development can be
found in some recent reviews on this subject.3942 Concern about the
risks of vaccination has also given fresh impetus to other methods, for
example, attempts to discover novel small molecule inhibitors of
dengue virus replication.43
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Immunology and Cell Biology