A specific and sensitive antigen capture assay for NS1 protein ...

Journal of Virological Methods 179 (2012) 8–16

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A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection Y.Z. Li a , D. Counor a , P. Lu a , G.D. Liang b , T.Q.H. Vu c , T.N. Phan d , T.K.L. Huynh c , G. Sun a , M. Grandadam e , S. Butrapet f , J.P. Lavergne g , M. Flamand h , Y.X. Yu i , T. Solomon f , P. Buchy j , V. Deubel a,∗ a

Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institute for Biological Sciences, 411 Hefei Road, Shanghai 200025, China Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), 155 Changbai Road Changping District, Beijing 102206, China c Institut Pasteur of Ho Chi Minh City, Ho Chi Minh City, 167 Pasteur Street, 8 Ward, 3 District, Ho Chi Minh City, Viet Nam d National Institute of Hygiene and Epidemiology, 1, Pho Yersin, Hanoi 10000, Viet Nam e Institut Pasteur, National Reference Center for Arboviruses, 25 Dr Roux Road, 25724 Paris cedex 15, France f University of Liverpool, Delby Street, Liverpool L69 3GA, UK g Institute of Biology and Chemistry of Proteins, 7, passage du Vercors, 69367 Lyon cedex 07, France h Institut Pasteur, Unit of Bunyaviridae, 25 Dr Roux Road, 75724 Paris cedex 15, France i National Institute for the Control of Pharmaceutical and Biological Products, Tiantan Xili 2# Chongwen Qu, Beijing 100050, China j Institut Pasteur in Cambodia, 5 Boulevard Monivong, Phnom Penh, Cambodia b

a b s t r a c t Article history: Received 5 February 2011 Received in revised form 4 June 2011 Accepted 8 June 2011 Available online 15 June 2011 Key words: Japanese encephalitis Flavivirus Genotype NS1 protein Monoclonal antibody Antigen-capture

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2 ng ml−1 NS1. Up to 1 ␮g ml−1 JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but 90% positivity for NS1 estimated by cytofluorimetry using a flavivirus cross-reactive anti-dengue 2 NS1 MAb (Fig. 1A). The NS1 protein from cell supernatants was purified by nickel affinity column and size exclusion column chromatography (Fig. 1B). The NS1 protein migration in Superdex 200 column showed three peaks, one >670 kDa that corresponded to aggregates or a multimer, one between 670 and 158 kDa that corresponded to the hexameric form of NS1, and one between 158 and 47 kDa that corresponded to the dimeric form (Fig. 1C). 3.2. Characterization of JEV anti-NS1 MAbs A panel of anti-NS1 MAbs was generated from mice immunized with the hexameric form of purified JEV NS1 protein. After fusion and selection procedures, hybridomas that produced antiNS1 MAbs were selected by testing cell supernatants by ELISA using NS1 antigen, and cloned. Two MAbs from a panel of 18 were selected because they showed the highest affinity for JEV NS1 hexamer tested by Biacore (data not shown). The affinity constant KD of MAbs 3E10 and 8F1 was 1.1 nM and 5 nM, respectively (Table 1). MAb mapping was performed using full-length NS11–352 , and Nterminal NS11–143 and C-terminal NS1244–352 fragments that were produced in S2 cells under denaturing (Western blotting of heated

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Y.Z. Li et al. / Journal of Virological Methods 179 (2012) 8–16

Fig. 1. Production and purification of soluble hexameric JEV NS1 protein in S2 Drosophila cell supernatant. (A) Flow cytometry of S2 cells and stable S2-NS11–352 cell clone, using the dengue flavivirus cross-reactive anti-NS1 MAb 17A12. Black line: S2 cells; blue line: S2-NS11–352 transfected cells; red line: S2-NS11–352 cell clone. (B) JEV NS1 protein purification steps. SDS-PAGE of S2-NS11–352 cell supernatant (1), after Ni-NTA chromatography (2) followed by purification on Superdex 200 column (3), indicated hexameric forms of NS1. MW: molecular weight. The arrow indicates the monomeric form of NS1 protein obtained after heating samples at 95 ◦ C. (C) Determination of the molecular weight of soluble extracellular NS1 protein by chromatography on Superdex 200 column. Arrows indicate the molecular weights of protein markers and NS1 hexamer. Samples obtained from the column were heated and examined by SDS-PAGE and stained with Coomassie blue. (For interpretation of references to color in this figure, the reader is referred to the web version of this article.)

NS1 protein) and non-denaturing (FACScan or indirect immunofluorescence) conditions of the proteins. Table 1 shows that MAb 3E10 recognized a conformational epitope and that MAb 8F1 reacted with a linear epitope of the NS1 protein that was located in the C-terminal fragment of the protein. The specificities of 3E10 and 8F1 in the flavivirus group were addressed by indirect immunofluorescence on Vero cells infected with different viruses. MAb 8F1 recognized NS1 in cells infected with JEV, WNV, MVEV, YFV and DEN-2V but not with tick-borne encephalitis virus, whereas 3E10 showed a positive fluorescence only with cells infected with JEV (Table 1). Immunofluorescence was negative for both MAbs with cells infected with the Chikungunya togavirus.

0.9979 (Fig. 2B). However, a cut-off value of 0.2 ng ml−1 was chosen, which gave two times the OD value obtained for negative controls using buffer instead of purified NS1 protein. The specificity of the JEV NS1 capture assay was tested against supernatants of Vero cells infected with JEV, WNV, MVE, YFV, DEN2V, and Usutu virus. Only supernatants of JEV-infected cells but not of other flaviviruses-infected cells were positive, which confirmed that the test specificity was restricted to JEV (Table 1). The specificity of the capture assay was tested against supernatants of Vero cells infected with the five genotypes of JEV. The assay was positive for genotypes I to IV, but negative for genotype V (data not shown). 3.4. Quantitation of viral RNA and NS1 protein during JEV replication in BHK and Neuro2A cells

3.3. Development of a capture assay for JEV NS1 protein An NS1 antigen-capture assay was optimized by testing different dilutions of MAb 8F1 coated on ELISA plates (capture antibody) followed by incubation with 1 ng purified NS1 hexamer. Captured NS1 was detected using different dilutions of biotin-labeled 3E10 MAb (revealing antibody). The best OD value was obtained for 250 ng/well 8F1 MAb and 400 ng ml−1 3E10 MAb conjugate (data not shown). An NS1 protein titration assay was established by using serial dilutions of purified NS1 hexamer from 100 to 0.1 ng ml−1 (Fig. 2A). A linear plot was observed from 0.1 to 1.6 ng ml−1 with an R2 of

The kinetics of viral RNA release and NS1 secretion were analyzed in BHK and Neuro 2a cells infected with SA14 JEV strain at an MOI of 0.1. Cells and supernatants were harvested at different times after infection. The onset of NS1 protein secretion was observed at 16 h after infection in both cell lines, at a concentration of 1–3 ng ml−1 , with a sharp increase from 24 to 48 h post-infection (Fig. 3A). NS1 (1 ␮g ml−1 and 670 ng ml−1 ) was detected at 48 h post-infection in supernatants of infected BHK and Neuro2A cells, respectively. Similar kinetics of virus release were observed in both infected cells, but this started about 8 h earlier than NS1 secretion (Fig. 3). Intracellular forms of NS1 were solubilized in RIPA buffer

Table 1 Characteristics of two MAbs chosen for specific JEV NS1-capture ELISA. MAb

Isotype

KD (nM)

3E10 8F1

IgG1 IgG1

1.1 5

a

ELISAa

WBb

FACScanc

IFA/NS1-capture ELISAd

JEV NS11-352 /NS1244-352

JEV NS11-352 /NS11-143 /NS1244-352

Cell surface/intracellular

JE

WN

MVE

DEN2

YF

USU

TBE

CHIK

+/− +/+

−/−/− +/−/+

+/+ +/+

+/+ +/+

−/− +/+

−/− +/+

−/− +/+

−/− −/−

NT/− NT/−

−/NT −/NT

−/NT −/NT

Mapping of 3E10 and 8F1 by direct ELISA. Purified NS11–352 or NS1244–352 antigens were coated on ELISA plates. Reactivity of MAbs for the S2-NS11–352 NS11–352 /NS11–143 /NS1244–352 . Cells lysates were tested by Western blotting (WB). c MAbs were incubated with S2-NS11–352 cells untreated or permeabilized (cell surface/intracellular) for fluorescence testing by flow cytometry (FACScan). d Immunofluorescence assay (IFA) and NS1-capture ELISA for testing 3E10 and 8F1 reactivity against Vero cells and supernatants infected with Japanese encephalitis (JE), West Nile (WN), Murray Valley encephalitis (MVE), dengue 2 (DEN2), yellow fever (YF), Usutu (USU), tick-borne encephalitis (TBE), Chikungunya (CHIK) viruses. b


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Fig. 2. Standard NS1 capture assay and linear plot. (A) NS1-capture ELISA consisted of plates coated with 8F1 MAb, incubated with purified NS1 hexamer (black dot) or with RIPA-buffer-treated NS1 (white dot), and revealed with 3E10 MAb conjugated to biotin followed by streptavidine–peroxidase complex. Tests were done in triplicate. (B) Segment of NS1 concentrations giving a linear plot of OD450 for calculation of the R2 for native and RIPA-buffer-treated NS1.

and quantitated by NS1 capture ELISA using a standard curve performed on purified NS1 hexamer after treatment with RIPA buffer. The R2 value of the treated NS1 titration (R2 = 0.9987) was close to that of the native NS1 (Fig. 3). Therefore, it was possible to compare the amounts of intra- and extracellular forms of NS1 from infected BHK and Neuro2A cells. For both cell types, the ratio of secreted to intracellular NS1 was close to 50% at 48 h post-infection (Fig. 3C).

3.5. Study of viral RNA, NS1 loads and of anti-NS1 antibodies in mice infected with JEV A time-course study of NS1 proteinemia and viremia, and of anti-NS1 IgM and IgG responses was carried out in JEV-SA14infected C3H/He mice (Table 2). Sera were collected in two to four mice for several days until day 15 after infection (the last day of mouse survival). Viral RNA copies were detected inconsistently and at low titers (from 0 to 66 × 104 RNA copies ml−1 ) (Table 2), in sera of mice from day 1 to day 15 after infection. The NS1 protein was detected by capture ELISA from day 5 to day 8 post-infection with an average concentration of NS1 from 1.5 to 6 ng ml−1 . There was no correlation between the presence of viral NS1 protein and viral RNA load (Table 3). Two mice were anti-NS1 IgM positive at day 5, two at day 6, and all mice were IgM-positive at day 7. Anti-NS1 IgG antibodies appeared only at day 8 post-infection in three mice and the titers increased until day 15 (Table 2). In an attempt to increase the number of samples positive for NS1-antigen, mouse sera containing IgM were pretreated with alkali pH 10.5 to dissociate NS1 from immune complexes, as described previously in mice

that contained both WNV NS1 and anti-WNV antibodies (Chung and Diamond, 2008). None of the NS1-negative samples became positive and no improvement in the titer of NS1-positive samples was observed after treatment (data not shown). To assess whether the presence of anti-NS1 antibodies affected the sensitivity of NS1 detection, NS1 protein was pre-incubated with anti-NS1 mouse IgM collected at days 6 and 7 after infection or with mouse IgM and IgG collected at days 12 and 13 after infection, and further treated or not with alkali. Preincubation with anti-sera containing IgM reduced by 2–4 the titer of NS1 calculated by NS1-capture ELISA, whereas preincubation with sera containing IgM and IgG reduced by 8 times its titer. Pretreatment of the immune complexes with alkali did not increase the sensitivity of the NS1-capture assay (data not shown).

3.6. NS1 capture assay in sera and CSF from patients infected with JEV A panel of sera and CSF of patients in China and Viet Nam, were obtained during national arbovirus surveillance programs. Patients showing clinical symptoms of febrile illness and encephalitis, were collected 1–8 days after the onset of the disease. All samples were tested for the presence of DENV and JEV by culture in C6/36 cell but remained negative after two passages. Samples showed previously a ratio >2 when tested for JEV versus DENV using IgM immunocapture assay, and were tested for JEV NS1-capture ELISA (Table 3). JEV NS1 antigen was detected in 10 out of 42 IgM-positive serum samples (23.8%) and in four out of 38 IgM-positive CSF samples (10.5%).

Fig. 3. NS1 and virus production in cells infected with JEV. (A) Titration of NS1 protein in JEV-infected BHK (black dots) or Neuro 2a (white dots) cell culture supernatants. (B) Titration of viral RNA copies in cell supernatants. (C) Percentage of cumulative amounts of secreted NS1 in total (cells plus supernatant) NS1. Cells were infected at an MOI of 0.1, and cell supernatants were harvested for JEV NS1 (NS1-capture ELISA) and RNA (real-time RT-PCR) titrations at different times post-infection. Three parallel wells were tested at each time point.

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Table 2 Viremia, NS1 antigenemia and antibody response in JEV-infected C3H/He mice. D1

M1

M2

M3