A sensitive epitope-blocking ELISA for the detection of Chikungunya virus-specific antibodies in patients

Highlights

• Developed epitope-blocking ELISA for detection of anti-CHIKV antibody response.

• Successful evaluation of diagnostic test with clinical serum samples.

• Diagnostic assay provides high sensitivity and specificity for clinical use.

• Utilization of virus-like particles as safe alternative to infectious virions.

• Rapid, cost effective, robust and simple-to-use diagnosis for screening.

Abstract

Chikungunya fever (CHIKF) has re-emerged as an arboviral disease that mimics clinical symptoms of other diseases such as dengue, malaria, as well as other alphavirus-related illnesses leading to problems with definitive diagnosis of the infection. Herein we describe the development and evaluation of a sensitive epitope-blocking ELISA (EB-ELISA) capable of specifically detecting anti-chikungunya virus (CHIKV) antibodies in clinical samples. The assay uses a monoclonal antibody (mAb) that binds an epitope on the E2 protein of CHIKV and does not exhibit cross-reactivity to other related alphaviruses. We also demonstrated the use of recombinant CHIK virus-like particles (VLPs) as a safe alternative antigen to infectious virions in the assay. Based on testing of 60 serum samples from patients in the acute or convalescent phase of CHIKV infection, the EB-ELISA provided us with 100% sensitivity, and exhibited 98.5% specificity when Ross River virus (RRV)- or Barmah Forest virus (BFV)-immune serum samples were included. This assay meets the public health demands of a rapid, robust, sensitive and specific, yet simple assay for specifically diagnosing CHIK-infections in humans.

Introduction

Chikungunya virus (CHIKV), the etiological agent of Chikungunya fever (CHIKF), belongs to the family Togaviridae and genus Alphavirus. CHIKV was first isolated in 1952 during an outbreak in Tanzania, East Africa (Robinson, 1955). Since then, CHIKV has caused sporadic epidemics of rheumatic disease though its prevalence has increased dramatically in recent years (Powers and Logue, 2007). Between 2004 and 2011, CHIKV was responsible for a massive outbreak at an estimated 1.4–6.5 million infections, with imported cases reported in over 40 countries (Suhrbier et al., 2012). Geographical expansion of the Aedes albopictus mosquito's distribution has allowed for the first autochthonous CHIKV infections in Italy and France, in 2007 and 2010, respectively (Rezza et al., 2007, Angelini et al., 2008, Grandadam et al., 2011). The increase in international travel has also seen the virus spread to previously unaffected countries such as the USA, the Caribbean and Australasia (Schwartz and Albert, 2010, Gibney et al., 2011, Viennet et al., 2013, Higgs, 2014, Mansuy et al., 2014).

CHIKF is characterized by an abrupt onset of fever, headache, myalgia, fatigue, nausea, rashes (usually maculopapular) and severe polyarthralgia, which can persist from weeks to months (Robinson, 1955, Brighton et al., 1983, Hoarau et al., 2010). The re-emergence of CHIKV has seen it be associated with severe disease manifestations and mortality, primarily in elderly patients with co-morbidities and the very young (Mavalankar et al., 2008, Economopoulou et al., 2009, Tandale et al., 2009, Jaffar-Bandjee et al., 2010). Furthermore, mother-to-child transmission was also observed with about half the infected neonates developing serious disease outcomes such as haemorrhage, disseminated intravascular coagulation and/or cardiac and neurological complications, with the latter often leading to permanent disabilities (Rampal et al., 2007, Gerardin et al., 2008, Suhrbier et al., 2012). The treatment of rheumatic disease caused by CHIKV currently involves the use of painkillers and/or non-steroidal anti-inflammatory drugs. Presently, there are no licensed human vaccines available although CHIKV vaccines are in development (Akahata et al., 2010, Wang et al., 2011, Brandler et al., 2013, Metz et al., 2013, Powers, 2014).

The clinical profile of CHIKF is similar to that of several other infections such as dengue fever, malaria and a host of closely-related arthralgic alphaviral diseases (Carey, 1971, Sergon et al., 2007, Suhrbier et al., 2012, Roth et al., 2014). Co-infection of CHIKV with dengue virus and other tropical arboviruses that cause non-specific symptoms similar to CHIKF have also been reported, highlighting the requirement of definitive diagnostic tests to assist clinicians with treatment options as well as to inaugurate appropriate public health measures (Vazeille et al., 2010, Kumar et al., 2012, Baba et al., 2013).

During early stages of the infection when antibody responses have not been generated, virus culture and/or PCR-based techniques are typically utilized for the detection of CHIKV. However, blood samples must be taken during the viraemic period, which typically lasts only 5–7 days (Edwards et al., 2007, Laurent et al., 2007, Santhosh et al., 2007). Moreover, this narrow window of opportunity for nucleic acid detection often starts prior to the onset of symptoms, thus further limiting the opportunity of obtaining viraemic patient samples for virus culture or PCR. Although the above-mentioned techniques are still considered gold standards for diagnosing CHIKV, both techniques require specialized facilities or equipment, as well as technical expertise to perform, which are too costly and impractical for widespread use.

In later phases of the illness (>1–2 weeks post-infection), IgM antibody-capture ELISAs (MAC-ELISAs) are the most commonly used tests for laboratory-based diagnoses. Other serological tests available include hemagglutination inhibition (HI), indirect ELISAs and virus-neutralization assays (Adesina and Odelola, 1991, Tiwari et al., 2009, Sam et al., 2011). However, most existing MAC-ELISA and HI tests are poorly established and lack credibility, in terms of specificity, due to the high possibility of false-positive results from cross-reactivity with other closely-related alphaviruses such as Ross river virus (RRV), Barmah Forest virus (BFV) and Sindbis virus (Niedrig et al., 2009, Rianthavorn et al., 2010, Blacksell et al., 2011, Kosasih et al., 2012, Reddy et al., 2012). Plaque-reduction virus-neutralization tests (PRNTs) are currently recommended by the world health organisation (WHO) for serological detection of CHIKV antibodies in human. However, these assays are not only labour-intensive, but also require access to bio-containment facilities due to the handling of infectious virus, rendering them impractical for rapid and high-throughput diagnostics.

According to the World Health Organisation, the ideal test should be rapid, specific, sensitive, cost-effective, user-friendly for lesser skilled personnel, and robust in different climatic conditions. More importantly, the test should also be equipment-free (in terms of dependence on electricity) and easily accessible to those who need it (Urdea et al., 2006). Herein we report the use of a monoclonal antibody (mAb) to the viral E2 protein and recombinant virus-like particles (VLPs), produced in insect cells (Metz et al., 2013), in an epitope-blocking ELISA (EB-ELISA) for the sensitive and specific detection of anti-CHIKV antibodies in human sera. The use of a CHIKV-specific mAb in the assay has allowed for the development of a highly-specific diagnostic test for CHIKV that should not produce false-positive results with closely-related alphaviruses, as well as other co-circulating arboviruses such as dengue virus. Furthermore, the utilization of stably-expressed non-infectious recombinant VLPs as the coating antigen will enable the assay to be performed safely without requirements of biosafety containment.