Expression, Purification, and Evaluation of Diagnostic Potential and Immunogenicity of a Recombinant NS3 Protein from All Serotypes of Dengue Virus

Dengue is one of the major public health concerns in the world. Since all the four serotypes are actively circulating in Mexico, there is a need to develop an efficient diagnosis system to improve case management of the patients. There exist few studies evaluating the use of the NS3 protein as a protective antigen against dengue virus (DENV). In this paper we show the expression of a recombinant NS3 protein from all serotypes of dengue virus (GST-DVNS3-1-4) and report a reliable “in-house detection system” for the diagnosis of dengue infection which was field-tested in a small village (Tezonapa) in the state of Veracruz, Mexico. The fusion proteins were immunogenic, inducing antibodies to be able to recognize to antigens up to a 1 : 3200 dilution. The purified proteins were used to develop an in-house detection system (ELISA) and were further tested with a panel of 239 serum samples. The in-house results were in excellent agreement with the commercial kits with κ = 0.934 ± 0.064 (95%  CI = 0.808–1.061), and κ = 0.872 ± 0.048 (95%  CI = 0.779–0.965) for IgM and IgG, respectively. The agreement between the NS1 antigen detection versus the rNS3 ELISA, κ = 0.837 ± 0.066 (95%  CI = 0.708–0.966), was very good. Thus, these results demonstrate that recombinant NS3 proteins have potential in early diagnosis of dengue infections.


Introduction
Dengue virus (DENV) infection in America, as in the rest of the world, is increasing dramatically. Currently, Mexico could be considered as an endemic region for dengue since the mosquito vector Aedes aegypti is present in more than 85% of the country [1]. Infection can lead to dengue fever (DF), a self-limiting febrile illness. A more severe form of the disease is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) with fatal consequences. Dengue consists of four, closely related but antigenically, distinct viral serotypes (DENV1-4) [2]. It is well documented that primary infection with one of the four serotypes confers long-lasting immunity to that specific serotype. However, secondary infection with a different serotype is associated with an increased risk of developing DHF where an antibodydependent enhancement (ADE) of infection is associated with the pathophysiological mechanisms of DHF [3,4].
The viral genome contains a single open reading frame that codes for a polyprotein of 3391 amino acids, which is processed into 10 individual proteins. Three of these proteins are structural (membrane (M), capsid (C), and envelope (E)) and 7 of them are nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [5][6][7]. The cleavage of this polyprotein, which represents an essential step for viral replication, is performed by host enzymes and the NS3 viral protease. The dengue non-structural 3 (NS3) is a multifunctional protein of approximately 69 kDa, involved in the polyprotein processing, RNA capping, and RNA replication. It contains a serine-protease domain, located at the N-terminal portion, and a helicase [8]. The dengue infection elicits different immune responses towards the viral proteins. Antibodies are generated mainly against the virus surface E protein and the secreted NS1 protein [9][10][11], while the majority of T-cell epitopes are concentrated within the NS3 protein, the main target for CD4 + and CD8 + T-cell response [11][12][13]. The E protein may also induce non-neutralizing antibodies involved in the phenomenon of antibody-dependent enhancement (ADE) of DENV infection, which can be associated to the occurrence of increased numbers of DHF in secondary infections [3,14]. Alternatively, some reports suggest the use of non-structural proteins for dengue vaccines to overcome such problem [15][16][17]. The NS1 is also highly immunogenic [18]; however antibodies against the NS1 may also cross-react with human proteins, which can be associated to some pathological effects of the dengue infection [19][20][21]. In contrast, there are only few studies evaluating the use of the NS3 protein as a protective antigen against DENV.
It is has been estimated that there are more than 3.6 billion people at risk of dengue infection with 36 million cases of dengue fever, more than 2 million cases of severe dengue, and more than 21,000 deaths occurring each year [22].
Since all the four serotypes are circulating in Mexico, there is a need to develop an efficient diagnosis system to improve case management of the patients. Until now, the incidence of dengue infection has been underestimated since most cases are not properly diagnosed, especially in small towns or villages where private or state laboratories for diagnosis are lacking [23]. According to this, early diagnosis during acute infection is critical to clinically manage severe disease and to identify potential outbreaks in a timely manner. Dengue infection diagnosis can be achieved by several assays such as RT-PCR [24], virus isolation [25], and NS1 antigen detection [19,26]. However, the enzymelinked immune assay (ELISA) has for a while, due to its ease, the routine diagnostic system for the dengue infection serological confirmation [27,28]. Different kits are commercially available, such as Panbio Dengue Duo IgM and IgG Rapid Cassette test kits and commercial Platelia Dengue NS1 antigen capture ELISA kit. Clearly, the availability of systems for the detection of dengue infections is a public health priority. Therefore, in this study, we show the expression of a recombinant NS3 protein from all four serotypes of dengue virus and we report a reliable "in-house detection system" for the diagnosis of dengue infection that was field-tested in a small village (Tezonapa) in the state of Veracruz, Mexico.

Immunization of Mice with GST-DVNS3-1, 2, 3, 4.
Female BALB/c mice (6-to 8-week old) were immunized by intraperitoneal route. All mice were maintained according to the recommendations by our Institutional Animal Care and Use Committee. The mice were immunized with one dose of 100 μg of antigen and two more with 50 μg. First immunizations were performed with the antigen emulsified in complete Freund's adjuvant (CFA), and re-immunizations at one-week intervals were performed with incomplete Freund's adjuvant (Gibco-BRL, Grand Island, NY, USA). The same schedule was used for control group, which received only GST plus adjuvant. At the end of the immunization scheme, animals were bled to obtain immune sera.

Study Area and Sample Collection.
The study was conducted in the municipalities of Tezonapa from the sanitary jurisdiction of Cordoba, in central Veracruz, located at latitude 18 • 36 north and longitude 96 • 41 west. A total of 10 rural localities were included: Caxapa, El Mirador, El Suspiro, Las Josefinas, La Joya, La Luna, Paraíso La Reforma, Rancho Nuevo, Raya Caracol, and San Agustín del Palmar. These villages are at an altitude of 80-700 m and are located at the junction of the coastal plains of Veracruz on the east and mountains of the Trans-Mexican volcanic belt on the west. In each village, research personnel provided general information on dengue virus and the project to households during open meetings organized in the rural medical units through the IMSS-Opportunities social program. Interested participants were given an appointment for their family at the medical unit for blood draws. On the appointment day, a written informed consent was obtained from each volunteer, and research personnel collected the blood samples in vacutainers tubes. Serum was separated by centrifugation at 1,200 ×g for 10 min and samples were stored at −70 • C until used.

Dengue Diagnostic.
A short questionnaire on knowledge of the disease and general clinical signs was also applied to the participants when blood samples were taken, with questions that included whether they have suffered fever higher than 37.5 • C, headache, retro-orbital and abdominal pain, vomiting, skin rash, nose or any other type of hemorrhage. A total of 239 serum samples were collected and analyzed for dengue virus infection using four different tests, including Panbio Dengue IgM and IgG Capture ELISA (Panbio Diagnostics, Brisbane, Australia), Platelia Dengue NS1 antigen capture ELISA (Bio-Rad) detection and inhouse system (anti-rNS3), and RT-PCR assay specific for a fragment of the protein E from dengue virus.

Dengue Diagnosis by an In-House System.
To test all serum samples, an indirect ELISA method was carried out as follows: 96-well plates were coated with 100 μL of carbonate buffer (pH 9.6) containing the antigen at concentration 2 μg/mL (pool of four recombinant NS3 proteins rNS3). After overnight 4 • C incubation, the plates were washed five times with PBS-T (0.05% Tween-20 in phosphate buffer solution), five minutes per wash, and blocked with 5% skimmed milk in PBS for 45 min at room temperature. Serum samples were serially diluted; the dilution that generated an OD value three times higher than that from negative samples was thoroughly used in the rest of the study. All serum samples were diluted 1 : 50, and 100 μL of each one was added to individual wells in triplicate and incubated for two hours at 37 • C. Further washing steps were conducted, and a peroxidase-labeled goat anti-human IgG antibody (Pierce, Rockford, IL, USA) was added at a 1 : 8,000 dilution in PBS/0.05% Tween 20 and incubated for 1 hour at room temperature. After eight washes, 100 μL of 2,2,-azino-bis(3-ethylbenzthiazoline)-6-sulphonic acid (Zymed, South San Francisco, CA, USA) was added as substrate and the reaction was allowed to proceed for 20 min at room temperature. The reaction was stopped with 2% sulfuric acid, and absorbance was read at 415 nm with an ELISA microplate reader (Multiscan MS; Labsystems, Vantaa, Finland). The cutoff (0.21) for this assay (at dilution 1 : 100) was established using the average obtained from a sample of 25 apparently healthy human sera plus two standard deviations (SDs). Positive samples were defined as samples with absorbance greater than two SDs above the mean of the negative control.

Data Analysis.
All proportion data are presented with a 95% confidence intervals (CIs). Using Fisher's exact tests we compared proportion data, and the kappa index was calculated when applicable. The relationship between age and seroprevalence rate was assessed by chi-square test and by regression analysis. All villages were georeferenced and a spatial database of serologic results was created in ArcView 3.2 (Environmental Systems Research Institute, Redlands, CA, USA) to produce seroprevalence maps.

Expression, Purification and Immunogenicity of Recombinant GST-DVNS3-1, GST-DVNS3-2, GST-DVNS3-3, and GST-DVNS3-4 Proteins.
Four DNA fragments were obtained from the cDNA encoding for NS3 domain protease (NS3pro185 sequence) from all serotypes of DENVs by PCR (Figure 1(a)). All fragments (NS3-DEN1/597 bp, NS3-DEN2/596 bp, NS3-DEN3/590 bp, NS3-DEN4/598 bp) (Figure 1(b)) were cloned into pCR2.1TOPO vector and subcloned again in correct open reading frame with orientation into pGEX-5X-1 expression vector ( Figure  1(a)). The expression of the recombinant proteins pGEX-NS3DEN1, pGEX-NS3DEN2, pGEX-NS3DEN-3, pGEX-NS3DEN4, and pGEX-5X-1 (parenteral plasmid) was evaluated in E. coli DH5α cells upon induction with IPTG at 37 • C. The proteins were purified from inclusion bodies. The amount of GST-fusion proteins present in the fractions corresponded to a yield between 1.8 and 5 mg/L culture depending on the recombinant protein; GST-NS3DEN3 protein expression was the lowest (1.8 mg/L). Figure 1(c) shows the fusion proteins obtained after 2 hours of induction and after the inclusion bodies were purified by solubilization with 2 M urea. After purification by affinity chromatography and analysis by SDS-PAGE, results revealed the expression of a novel protein of ∼49 kDa (Figure 1(c), lane 1-4) and expression of GST as a 27-kDa protein (Figure 1(c), lane 5). They were detected by western blotting using a polyclonal antibody against GST proteins, and all of them had the predicted weight.
In order to establish that these purified GST dengue proteins can induce humoral immune response, groups of 5 BALB/c mice were immunized with recombinant proteins. Serum samples were analyzed at the end of the immunization scheme. The fusion proteins were immunogenic, inducing antibodies able to recognize antigens up to 1 : 3200 dilution (data not shown).

Discussion
Dengue infection is a growing public health concern in endemic areas all over the world. Hyperendemic geographical areas have been defined as those with continuous presence of multiple viral serotypes and competent vectors, and a large population of susceptible hosts, as it seems to be the case for Mexico [1]. Despite the importance of the DENVs as emerging pathogens, diagnostic tests remain inadequate for efficient and accurate identification of DENV infection. Mexico, currently, has a network of public health laboratories which consists of 30 laboratories that perform confirmation diagnosis of dengue by using immunoassay techniques for detection of viral antigen NS1, IgM antibody, or IgG, depending on the time evolution of the disease when the patient seeks medical care. It is well recognized that there is a high level of underreporting of cases of dengue even in areas where there is an adequate surveillance system. It is expected that the level of underreporting in dispersed rural areas and with poor access to health services is even higher [35]. Our study focused on diagnosis of dengue virus infection as well as on the presence of antibodies in rural areas of central Veracruz state. Current dengue diagnostic methods have a number of serious limitations. The gold standard for diagnosis of acute DENV infection is viral isolation, but the procedure is costly (US$39.10), time consuming, and technically difficult to perform. Reverse transcriptase PCR (RT-PCR) has been widely adopted as an alternative for viral isolation in the diagnosis of acute infection, but PCR is technically intensive and expensive (US$136.67), and its sensitivity varies from 80 to 90% based on primer sets. An IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is useful primarily for diagnosing dengue infection in the late acute or early convalescent phase of the illness but is often insensitive for early-acute-phase infections [36]. The distinction between primary and secondary infections is currently assessed by measuring IgM and IgG responses to dengue antigens in paired serum samples taken from a febrile patient in the acute stage of disease and after convalescence [37]. In order to set up a rapid and reliable diagnosis, some laboratories in Mexico use the Panbio Dengue Duo IgM and IgG Rapid Cassette test kits or the ELISA Dengue IgM capture kits. However, it is sometimes difficult to have access to these kits due to stock shortage in the market, problems to introduce foreign products, and of course, high cost. Therefore it is necessary to find alternatives for the diagnosis of DENV during the acute phase of disease, because diagnostic tests remain inadequate for efficient and accurate identification of DENV infection. Similarly, very good agreement is observed when comparing the NS1 antigen detection versus rNS3 ELISA. Nonstructural protein 1 (NS1) of DENV has been shown previously to be useful as a tool for the diagnosis of acute dengue infections. NS1 has been detected in the sera of DENV-infected patients as early as the first day of the symptoms and up to 18 days [38]. Our results showed that all samples that were positive for antigen detection NS1 were also positive for the presence of antibodies against the recombinant NS3 protein. These results suggest that the use of ELISA with recombinant NS3 protein may be an alternative method for serological analysis of dengue virus in the acute phase. NS3 protein plays a predominant role in the pathogenesis of the disease and together with its role during the processing of the precursor of the viral polyprotein makes it an interesting protein for evaluating host responses. Some authors have reported a significant antibody response to NS3 in both primary and secondary infections [39][40][41]. However, Valdes et al., 2000 [40], showed a significant antibody response in secondary cases when the infecting serotype was DEN2. In this case, in-house detection system used an anti-human IgG antibody as second antibody, which allowed to suggest that antibodies present in the sera of patients were IgG but not IgM antibodies. This is consistent with the findings from Valdes et al. [40], as we found that IgG antibodies for a secondary response and virus serotyping results show that the major infecting serotype was DEN2. The conclusion that the use of ELISA with recombinant NS3 protein may be an alternative method for serological analysis of dengue virus in the acute phase results from the fact that there is an excellent concordance when comparing the NS1 antigen detection versus rNS3 ELISA. It has been reported that dengue is transmitted in rural areas and currently has become condition of urban nature. In addition, migrations of population favor a permanent risk for the spread of dengue-endemic areas.
The characteristics of an "ideal" dengue diagnostic test depend on the purpose for which the test will be used; in case of early diagnosis the dengue test should distinguish between dengue and other diseases with similar clinical aspects (such as malaria, leptospirosis, and typhoid), highly sensitive during the acute stage of infection, provides rapid results, inexpensive, easy to use, and stable at temperatures greater than 30 • C for use in rural areas, if necessary [42]. Additionally to this, the study of the predictive ability of a diagnostic test on the in-house dengue system was observed with a good specificity and sensitivity and provides rapid results (3 hours); moreover it is less expensive than commercial tests available in the market. Serological tests have an intermediate price compared to molecular tests. In fact, when quoting a new test, the laboratories indicated that it should be considered a 4-7-fold increase versus the reagent price. In this sense, the cost assessment of "in-house" ELISA and commercial ELISA kits showed that "in-house" ELISA proved to be a cheaper kit than the commercial ones, with a cost of US$8.20, while other kits have higher costs as it is showed next: NS1 antigen detection (US$18.80), IgM (US$22.62), and IgG capture ELISA (US$24.04). This share reflects the actual costs of each test method based on the actual cost of reagents, laboratory supplies, stationery, personnel, and indirect costs as electricity, water, depreciation of equipment, and so forth.
Finally, the identification of the circulation of more than one serotype in the municipality of Tezonapa (DEN1 and DEN2) is a warning sign because it may favor the increase of severe clinical forms of dengue due to the presence of secondary infection by different serotypes, promoted by the phenomenon of increased antibody-dependent infection. We propose the use of recombinant NS3 protein as a detection system as a viable alternative in Mexico, when commercial kits are not available, at least in terms of a first screening. Even if this system needs to be improved by increasing the number of samples, for example, specially from asymptomatic individuals from the endemic regions in Mexico, to increase its accuracy in the dengue virus diagnosis, it is important to keep working on the development of reliable diagnostic tools in order to establish an efficient surveillance system in dengue endemic areas.