Date Published: November 13, 2015
Publisher: Public Library of Science
Author(s): Alexandra Rockstroh, Luisa Barzon, Monia Pacenti, Giorgio Palù, Matthias Niedrig, Sebastian Ulbert, David W.C. Beasley. http://doi.org/10.1371/journal.pntd.0004218
Abstract: Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.
Partial Text: Dengue virus (DENV) is a mosquito-transmitted pathogen of the family Flaviviridae, a group of small, enveloped and positive stranded RNA-viruses. Besides DENV there are several other human pathogenic vector-borne flaviviruses, such as yellow fever virus (YFV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) or Japanese encephalitis virus (JEV) . DENV is endemic to over a hundred tropical and subtropical countries worldwide, and the numbers of annual infections are strongly increasing with a current estimate of 400 million . High fever is the most common clinical symptom of a DENV-infection, but also severe complications are observed, such as dengue shock syndrome (DHS) or dengue haemorrhagic fever (DHF). Approx. half a million hospitalizations and several thousands of fatalities are caused by DENV every year [3,4]. Dengue viruses include four major distinct serotypes, named DENV-1 to DENV-4, and survival of an infection with one of these serotypes leads to a lifelong immunity to this serotype, but not to the others . In parallel to the increasing distribution of its vector, mosquitos from the genus Aedes, DENV is emerging or re-emerging in several areas including Europe and North America. In Europe local transmission of the virus has been demonstrated in France and Croatia in 2010 [6,7]. In addition, a DENV-outbreak occurred in Madeira in 2012 and resulted in over 2000 cases and transportation of the virus into several other European countries .During the acute phase of infection, Dengue can be diagnosed by directly detecting viral RNA or the non-structural protein 1 (NS1), which is secreted by infected cells. About five days after onset of symptoms, these direct infection markers start to decrease in blood and DENV-IgM antibodies appear, followed by IgG a couple of days later. Therefore, except during the acute phase, DENV-infections are usually diagnosed by antibody-measurements, and several serological test systems are available [reviewed in 9,10]. Whereas IgM-detection can indicate recent primary infections, IgG-measurements are useful for detection of secondary infections, where IgM responses usually remain lower, and for serological surveillance activities. Acute dengue infections can be confirmed via a rise in IgM or IgG levels in paired samples . One of the major problems in antibody-based diagnosis of dengue is the similarity of structural proteins of different flaviviruses which leads to cross-reactive antibodies and false positive test results [11–13]. Especially in areas where several flaviviruses co-circulate or in the context of vaccination against e.g. YFV or TBEV, this is of concern [1,14]. As the currently available tests cannot exclude flavivirus cross-reactivity, positive test results have to be confirmed by virus neutralization tests, which are time consuming and require BSL-3 laboratories. The E (envelope) protein is a major target of the human antibody response during DENV infections and is used in most available tests . Cross-reactive antibodies target mainly the highly conserved fusion loop (FL) domain of the E protein which is involved in fusion of the viral and cellular membranes [16–19]. As a consequence, the insertion of mutations into the FL leads to a decrease in binding of cross-reactive antibodies, which has been employed to develop diagnostic methods on the basis of WNV- or JEV- virus-like particles (VLPs) [20–22]. Alternatively, other recombinant antigens than the E protein, such as NS1, have been used to differentiate flavivirus antibodies via titer-determinations .
To enhance specificity of serological DENV diagnosis we inserted 4 amino acid point mutations into the conserved fusion loop (FL) domain and an adjacent loop domain of DENV wildtype (wt) E proteins, yielding quadruple mutants for DENV serotypes 1 to 4 (DENV 1–4 Equad-proteins, Fig 1). The mutant proteins as well as the DENV-2 wt E-protein were overexpressed in Drosophila S2 cells and secreted into the culture´s supernatant. After purification by immobilized metal affinity chromatography several unspecific proteins were co-purified. Therefore a second purification step using size exclusion chromatography was performed to eliminate the visible unspecific bands (Fig 2A).
A challenge for current serological dengue diagnosis is the high degree of cross-reactivity between antibodies produced during infections with related flaviviruses [27–29], leading to false positive results in currently available serological test systems [11,12]. Especially in areas with different co-circulating flaviviruses, this is of concern [1,14]. Here, we present insect-cell derived recombinant DENV E proteins bearing mutations in the conserved FL domain to enhance the specificity of serological DENV diagnosis. When using the wild type E protein from serotype 2 (which is the basis for several available test systems) we found all the DENV-positive sera displayed high signals (Fig 4A). However, the cross-reactivity problem was also confirmed, as the signals obtained with many WNV- and TBEV positive samples were in the range of DENV-signals, although the mean intensities were lower (Figs 3E and 4A). As a consequence, several sera would be misdiagnosed as false-positive, which was indeed observed when a commercial assay was used (Fig 5).