T cell responses and dengue haemorrhagic fever

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Gavin Screaton and Juthathip Mongkolsapaya

Department of Immunology, Hammersmith Hospital, Imperial College, Du Cane Road, London W12 0NN, UK

Abstract. The enhancement of severe disease upon secondary infection makes dengue almost unique among infectious pathogens and presents a serious challenge to vaccine design. Several key observations have been made which shed light onto this phenomenon particularly that antibodies can enhance Fc receptor-dependent uptake of virus into macrophages thereby increasing virus replication. Furthermore there seems to be a relationship between the peak virus load and disease severity. However, a second key feature of dengue is that the life-threatening symptoms do not correlate with the period of high viraemia; instead they occur at a time when the virus load is in steep decline. The coincidence of severe disease manifestations with defervescence and virus control suggests that the symptoms may be a consequence of the immune response to the virus rather than virus induced cytopathology. One of the key elements in the immune response to viruses are T cells which can both secrete a host of inflammatory cytokines and also be directly cytotoxic to infected cells. There are a number of experimental models of T cell-induced immunopathology including in responses to viruses. Particularly interesting in this respect are models of RSV-induced immunopathology, which have direct relevance to vaccine design as a formalin-inactivated vaccine to RSV actually enhanced disease in children when they became naturally infected with RSV, an echo of the disease enhancement seen in dengue. We will present an analysis of CD8 + T cell responses to a number of novel T cell epitopes during dengue infection and also analyse the function and cytokine secretion of these cells. We suggest that an exaggerated and partially misdirected T cell response seen in secondary dengue infection may be part of the complex series of events leading to dengue haemorrhagic fever and shock.

2006 New treatment strategies for dengue and other flaviviral diseases. Wiley, Chichester (Novartis Foundation Symposium 277) p 164—176

Infection by dengue virus has become a major public health threat in tropical and sub tropical countries. The virus belongs to the family flaviviridae and circulates as four major serotypes. The virus is still evolving and the serotypes differ in sequence by around 30%. Dengue is transmitted to humans following a bite from an infected mosquito, usually Aedes aegypti.

Following the bite there is an incubation period of 3—8 days which is followed by a symptomatic phase, although school based serological surveys suggest that

50% or more of infections are asymptomatic (Endy et al 2002). Symptoms, which range from a mild undifferentiated fever to more severe fever, headache, muscle, joint and bone pains accompanied later on in the illness by a maculopapular rash. This more severe clinical syndrome is classified as dengue fever and may be accompanied by thrombocytopaenia leucopaenia and petechial haemorrhage (World Health Organization 2002).

More severe manifestations of the disease are classified as dengue haemorrhagic fever (DHF). This is a potentially life threatening condition with up to 20% mortality without expert medical care. DHF is characterised by plasma leakage and thrombocytopaenia and can be divided into four clinical grades with increasing severity of bleeding, vascular leakage, hypovolaemia and shock.

One highly characteristic and interesting feature of DHF is that the severe symptoms of bleeding and circulatory collapse seem to occur coincidentally with the time of defervescence, the point at which the fever subsides. Before this and for the 2—7 days of fever these patients will resemble cases of dengue fever.

The mainstay of treatment for DHF is careful management of fluid status with replacement with isotonic saline, colloidal plasma expanders or blood where required. With careful monitoring and attention to fluid balance the mortality can be reduced to substantially below 1%.

Despite this dengue infection still remains a major public health issue in a number of tropical and subtropical countries. About 2.5 billion people are at risk of dengue infection and there are estimated to be 50—100 million infections annually. The disease occurs in an epidemic fashion and therefore puts a huge strain on health care services; in Thailand for instance there are around 100 000 cases of DHF annually.

The incidence of dengue infection has increased rapidly since the Second World War. DHF which was rare or absent in many parts of the world has also become much more frequent. Many explanations of this huge expansion of disease have been put forward including a drive toward urbanisation and travel allowing rapid dissemination and cocirculation of viral strains (Mackenzie et al 2004).

Careful epidemiological analyses often in island populations have yielded very valuable information about the pathogenesis of severe dengue infection or DHF. Some of the best of such evidence has come from Cuba (Guzman et al 2000). In 1977 there was an epidemic of dengue fever cased by the DENV-1 serotype of virus. The majority of the population was dengue naïve and interestingly during this outbreak there were few cases of severe disease. Infection with the DENV-1 serotype reduced in subsequent years until in 1981 there was a second epidemic caused by the DENV-2 serotype. On this occasion the spectrum of disease was markedly different; there were nearly 350 000 cases of dengue fever, over 10 000 cases of DHF and around 150 deaths.

This and a number of similar studies have shown that the risk of developing DHF is much higher in people who have been previously exposed to one serotype of dengue and who are subsequently exposed to a secondary or sequential infection with a virus of different serotype (Sangkawibha et al 1984). In this case it therefore appears that pre-existing immunity or memory for the previous infection can actually potentiate disease. A further epidemic of DENV-2 in Cuba in 1997 demonstrates that the risk from previous infection (DENV-1 1977) can last for 20 years or more.

Antibody dependent enhancement has been suggested as a mechanism of disease potentiation (Halstead & O'Rourke 1977). This was proposed by Scott Halstead in 1977 and was based on similar observations on the related flavivirus tick-borne encephalitis (Phillpotts et al 1985). It is proposed that following a primary dengue infection an antibody response is mounted to envelope proteins on the infecting virus. Since different viral serotypes will differ by around 30% in sequence, antibodies directed to one serotype may not confer complete neutralising protection to another serotype. Following primary infection as the titre of these antibodies falls it is proposed that there becomes a point where the antibodies may bind to virus and rather than neutralize infection completely actually target uptake of opsonized virus by Fc-receptor bearing cells, principally macrophages thereby driving higher viral replication.

This phenomenon can be demonstrated in vitro and there is some evidence that it can operate in vivo (Halstead 1979). Indeed, antibody-dependent enhancement has been invoked to explain a small peak in severe dengue infection in children below the age of one year which can occur during a primary exposure and is proposed to be driven by the fall in titre of maternally acquired anti-dengue antibodies which occurs with age (Halstead et al 2002).

Recently, viral loads have been measured throughout the course of infection using either virus culture or reverse transcriptase PCR. There appears to be a correlation between higher viral titres and more severe disease (Vaughn et al 2000). Interestingly however, the virus load falls precipitously at the time that the fever remits. At this point, when symptoms peak, virus titre is low or undetectable. The lack of correlation between virus load and severe symptoms has led some to speculate that the sequelae of severe dengue infection may be driven more by the immune response to the virus than virus load per se.

Several groups including our own have measured the levels of inflammatory cytokines during dengue infection. Many of these are raised and in some the peak in their levels coincides with the onset of severe symptoms. High levels of tumour necrosis factor (TNF)a, interferon (IFN)y, interleukin (IL)10, IL6, etc. have been recorded and many of these can be the products of activated T cells (Rothman & Ennis 1999). These findings have led some to suggest that it is T cells that cause the damage and vascular leak characteristic of dengue either by causing direct cytotoxic tissue damage or by producing a variety of inflammatory cytokines.

Like B cells, T cells belong to the acquired immune system in that the antigen receptor is not germline encoded as with molecules characteristic of the innate immune system but instead constructed following a series of genomic recombinations which occur in the thymus. The T cell receptor is a non-covalently linked heterodimer, the major form consisting of a pairing of a and p chains whilst a minor form consists of a pairing of y and 8 chains (Janeway et al 2001). T cell receptors recognise short antigenic peptides which are bound in the antigen binding groove of MHC molecules. MHC-I presents 8-11mer peptides which are derived predominantly from intracellularly produced proteins to cytotoxic T cells bearing the CD8 co-receptor. MHC-II presents slightly longer, predominantly endocytosed exogenous antigen to helper T cells bearing the CD4 co-receptor.

To study T cell responses in detail it is necessary to define the sequence of the antigenic peptides from dengue presented to T cells. Since MHC is highly polymorphic there will likely be a large number of dengue peptides which can bind to different MHC molecules. Several T cell epitopes for dengue have been previously described but these are mostly restricted by Western MHC types which make their utility to examine T cell responses in endemic areas such as SE Asia limited.

To overcome this problem, we undertook a systematic search for dengue T cell epitopes using an overlapping peptide approach (Altfeld et al 2000). MHC molecules bind linear peptides between 8-11 amino acids in length and the peptide binding groove can accommodate, although with less efficiency, short peptide extensions at either end. Therefore, a nested set of peptides 15-20 amino acids in length and overlapping by 10-11 amino acid will contain all possible 10-11 linear amino acid sequences in effect covering all possible antigens and can be used for antigen discovery.

The presence of responding T cells can then be assessed by culturing peripheral blood mononuclear cells from dengue immune individuals with pools of these overlapping peptides. If responding T cells are present then they will be activated by peptide and this activation can then be read by assaying cytokine production from the activated T cells. We used the IFNy Elispot assay for this purpose where a sandwich ELISA for IFNy allows the enumeration of the responding T cells.

Using this assay we were able to discover a number of T cell epitopes for both CD8 + and CD4 + positive T cells. We were particularly interested in those epitopes which were restricted by commonly expressed MHC-I alleles in SE Asia such as A11, A24 and A33. An immunodominant epitope from the NS3 protein GTSG-SPIIDKK which was restricted by HLA-A11 was found and studied in detail (Mongkolsapaya et al 2003). T cell responses to this epitope were examined in a cohort of children admitted to hospital in Khon Kaen in North East Thailand.

Since dengue virus seroytypes show about 30% amino acid difference we searched sequence databases to ascertain whether differences were found in this A11 epitope and found six variants of the epitope (Table 1) which were expressed by the variant viruses. Responses to these variant epitopes were analysed in the patients' samples using both Elispot and MHC tetramer analysis using fluorescence-activated cell sorting (FACS) (McMichael & O'Callaghan 1998).

All of the patients in this study were suffering from secondary dengue infections and they showed diverse T cell responses to the variant peptide epitopes derived from the different dengue serotypes. During the acute illness dengue-specific T cells were highly activated and almost all also showed signs of cell proliferation and apoptosis. There was also a correlation between the magnitude of the T cell response and the severity of the dengue illness (Fig. 1).

When we looked at the fine specificity of the T cell response we found paradoxically that many of the T cells had a relatively low affinity for the currently infecting viral serotype and showed higher affinity for serotypes which we presume had been encountered before (Fig. 2).

This phenomenon whereby a response to a variant of a previously encountered epitope is constructed mainly from memory T or B cells rather than being generated de novo by fresh priming is termed original antigenic sin (Fazekas de St & Webster 1966b,a, McMichael 1998). Original antigenic sin has the advantage that a cross-reactive response can be rapidly recalled from memory but has the disadvantage that the response may not be optimal and may contain some lower affinity T cells. In some experimental circumstances such as murine infection with variants of the lymphocytic choriomeningitis virus original antigenic sin has been shown to be detrimental leading to slower clearance of virus in a secondary as opposed to primary infection (Klenerman & Zinkernagel 1998).

In summary, it seems likely that the increased severity of a secondary or sequential dengue infection is immunologically driven and multifactorial. Antibody

TABLE 1 A list of GTS variants obtained from dengue sequences published in Genbank

Variant

Sequence

Number of sequences found/total

Den 1.1

GTSGSPIVNRE

5/5

Den 2.1

GTSGSPIIDKK

29/39

Den 2.2

GTSGSPIVDKK

6/39

Den 2.3

GTSGSPIVDRK

3/39

Den 2.4

GTSGSPIADKK

1/39

Den 3.1

GTSGSPIINRE

2/2

Den 4.1

GTSGSPIINRK

2/2

Normal

DHFI/II

DHF III

FIG. 1. Frequency of dengue-specific CD8 + T cells at convalescent day 4 from patients with different disease severities; dengue fever (DF), dengue haemorrhagic fever grades I and II (DHF I/II) and DHF grade III compared with normal healthy dengue-immune individuals (normal).

Current: DENV-2 infection Previous: DENV-1 infection

FIG. 2. Secondary dengue infection showing the original antigenic sin phenomenon. PBMCs from DENV-2-infected patients were simultaneously stained with the GTS tetramers from DENV-1 (D1) and DENV-2 (D2) and analysed by Flow cytometry. A substantial number of T cells are of higher affinity for the previously encountered DENV-1 as opposed to the currently infecting virus DENV-2.

FIG. 2. Secondary dengue infection showing the original antigenic sin phenomenon. PBMCs from DENV-2-infected patients were simultaneously stained with the GTS tetramers from DENV-1 (D1) and DENV-2 (D2) and analysed by Flow cytometry. A substantial number of T cells are of higher affinity for the previously encountered DENV-1 as opposed to the currently infecting virus DENV-2.

dependent enhancement may drive virus internalisation into macrophages and enhance viral replication. At the same time original antigenic sin may initially delay an effective high affinity T cell response to the virus allowing further viral replication whilst a high affinity T cell response is generated. This will lead to a dangerous situation where there is finally the collision of a large number of T cells with a high antigen load. This will lead to massive T cell activation which will cause both direct T cell mediated cytotoxicity and the release of a variety of inflammatory cytokines. These in turn will lead to tissue damage and the syndrome of vascular leak which occurs coincidentally with viral clearance in DHF.

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