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Rev. Inst. Med. trop. S. Paulo vol.44 no.3 São Paulo 2002
The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity .
KEYWORDS: Rabies virus; Flow cytometry; Neutralizing antibodies.
The determination of an antibody response after immunization against rabies is an accepteble index of the efficacy of vaccine and successful treatment in both animals and humans. Among the different antibodies elicited after immunization, those specific for the virus glycoprotein (neutralizing antibodies) are considered the most important to provide protection (WIKTOR et al., 1973). Several in-vitro tests have been developed to assess neutralizing antibodies, including those based on fluorescent focus inhibition (SMITH et al., 1973; ZALAN et al., 1979) which are the most widely used. The Brazilian National Reference Institute for rabies is Pasteur Institute (São Paulo), which uses the simplified fluorescent inhibition test - SFIMT - (FAVORETTO et al., 1993) in its serological routine. In addition, several variations of Enzyme-linked immunosorbent assay (ELISA) have also been proposed by several authors as a possible alternative to cell-culture tests (ATANASIU et al., 1977; ELMGREN & WANDELER, 1996; ESTERHUYSEN et al., 1995; NICHOLSON & PRESTAGE, 1982; PERRIN et al., 1986; PIZA et al., 1999). Flow Cytometry, a methodological analysis approach based upon refracted light, has been used for detecting antibodies to different viruses such as HCMV (McHUGH et al., 1986 and 1988), HSV (SCILLIAN et al., 1989) and HIV (SLIGH et al., 1989). We have recently reported the use of Flow Cytometer for rabies virus detection (BORDIGNON et al., 2002) and the present study describes the standardization of a Flow Cytometry technique for detection and quantification of rabies VNA. Results are compared to those obtained by SFIMT and ELISA.
MATERIALS AND METHODS
CELL CULTURE: BHK-21 cells (C-13) (ATCC CCL10) were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (Sigma #D-8900), supplemented with 2 mM glutamine, 10% (v/v) fetal calf serum (Sigma #F-2442) and 1 X antibiotic/antimycotic solution (Sigma #A-5955) at 37ºC. Confluent cell monolayers were trypsinized with 0.25% Trypsin-EDTA solution (Sigma #T-4049).
VIRUS: The fixed rabies virus (PV) was supplied by the Rabies Diagnosis Laboratory of the Instituto Pasteur, São Paulo, Brazil. The work dilution of virus was established by incubating 5 x 104 cells in 100 ml with a serial two-fold dilutions of the virus suspension (50 ml). One cell-culture infective dose (CCID50) was then established as being the reciprocal of the dilution (1:80) which infected about 50% of the cells. One CCDI50 was used in all experiments.
SERUM SAMPLES: The 104 heat-inactivated dog serum samples used in this study were kindly supplied by the Serology Section of Instituto Pasteur (São Paulo, Brazil). An International Reference Serum (Copenhagen, Denmark) containing 30 IU/ml was used in parallel in all experiments.
ELISA: The immunoassay used was that previously described by PIZA et al. (1999), adapted by RODRIGUES da SILVA et al. (2000), excepting for the dog serum samples and the anti-Dog IgG conjugate (Sigma #A6792) which were diluted at 1:200 and 1:10,000, respectively. Antibody titres were expressed as equivalent units (EU/ml).
FLUORESCENCE INHIBITION MICROTEST: Neutralizing antibodies were measured by the simplified fluorescence inhibition microtest - SFIMT (FAVORETTO et al., 1993), currently used at Pasteur Institute of São Paulo. Serum titers (expressed in IU/ml) were obtained by comparison of the results of unknown sera and standard serum, as described by SMITH et al., 1996.
Virus neutralization in cell culture for FC analysis: One hundred microliters of an optimal pre-determined serum sample dilution (1:25) were added to 24-well plates and mixed with 1 CCDI50 of PV virus (100 ml). A titration of the International Reference Serum, pre-diluted to contain 20 IU/ml, was always performed in parallel. Serum samples and virus were then incubated for 45 min (37ºC, 5% CO2) and 5 x 105 BHK-21 cells/well were then added. Plates were incubated for additonal 24 h. Cell monolayers were disrupted by incubating with 500 ml of trypsin-EDTA solution for 5 min. The detached cells were recovered and resuspended in 2.0 ml MEM supplemented with 10% FCS. The cells were washed twice with PBS containing 0.05% Tween-20 and centrifuged at 3,000 rpm in a bench centrifuge at room temperature.
Cell fixation and staining: After detached and washed, cells were resuspended with 1 ml solution of 4% (v/v) solution of p-formaldehyde and 1 ml of a 1:10 FACS lysis fluid (Beckton Dickinson #349202) dilution in water. After 10 min incubation at room temperature, the cells were washed twice again as described before. The pellet from the last wash process was resuspended in 100 ml of a 1:20 dilution of rabies virus nucleocapsid protein-specific antibody conjugate solution (Sanofi, France) in PBS containing 0.2% of Evan Blue, and incubated for 45 min in the dark at 37ºC. After this last incubation, the cells were centrifuged and washed as before. The final pellet was resuspended in 1 ml of ISOTON IIâ (Coulter) and stocked at 4ºC (no longer than 24h) until flow cytometry analysis. Uninfected cells and infected cells incubated with negative serum, submitted to the same procedure as above were used as negative and positive control, respectively.
Flow cytometry analysis: A Beckton Dickinson FACSCaliburâ flow cytometer equipped with a 15 mW 488nm air-cooled argon-ion laser was used to analyze the cell infection kinetics. The forward scatter (FSC) detector was set at E00 with a threshold value of 200, and the side scatter (SSC) at 370 V. An area containing all cellular events was defined as R1. A total of at least 10,000 events were recorded for each sample at R1 area. For fluorescence analysis, the intensity of the green fluorescence (FITC - FL1 detection) was obtained from the R1 area. The FL1 was set at 325 V. Such voltage placed the negative control cells bellow 101, containing at least 97% of these cells. Markers M1, between 100 and 101, and M2, between 101 and 104, were determined. M2 values define the percentage of cells considered as infected (positive for intracellular viral particles).
Calculations of VNA titers: The maximum rate of infection (100%) was obtained when cell suspension were infected with rabies virus with a negative control serum. Based upon the rates of infection obtained with the serial dilutions of reference serum, a standard curve was drawn correlating the percentage of infection inhibition and the contents of IU/ml of the serum dilutions, as shown in Fig. 1. The values of EU/ml of the other 104 serum samples were obtained by extrapolation.
STATISTICAL ANALYSIS: Results obtained by the different techniques were compared by the non-parametric Wilcoxon rank test and Spearman correlation test.
Optimization of technical parameters: The VNA titers of 104 dog serum samples were evaluated by flow cytometry and results were compared to those obtained from SFIMT and ELISA. A checkerboard titration employing a negative and the positive reference sera was initially performed to determine the best number of cells/well, the best dilution of sera and virus concentration to be used in flow cytometry analysis. The best results were obtained employing 5 x 105 cells/well, 1:25 serum samples dilutions and 1 CCDI50 (1:80) of rabies virus. Even minute modifications of those parameters profoundly affected the final results (data not shown).
Standard curve of infection inhibition: Fig. 1 shows the percentage of inhibition of viral infection of BHK-21 brought about by the decreasing dilutions (or increasing IU/ml-concentration) of the reference standard serum in four independent experiments. Serial twofold dilutions of reference serum ranging from undiluted up to 1:16 (corresponding 30 to 1.88 IU/ml, respectively) completely abrogated viral infection. Even when the reference serum was diluted to contain 0.16 IU/ml, a significant reduction (20%) of viral infection could be observed.
Correlation between FC X SFIMT and FC X ELISA: In contrast to the reference serum which was used in a serial two-fold dilutions in all experiments, dog serum samples were always tested in a single dilution, 1:25. The VNA titer of each serum sample, obtained by flow cytometry, was compared to conventional techniques. As shown in Fig. 2a, Flow cytometry and SFIMT results showed a high statistical correlation (Wilcoxon rank test: p < 0.001; Spearman correlation coefficient r = 0.7734; p < 0.001).
When VNA titers obtained by flow cytometry was compared to those obtained by ELISA, there was a lower statistical correlation between the results (Wilcoxon rank test: p < 0.001; Spearman correlation coefficient r = 0.5928; p < 0.001) as shown in Fig. 2b.
The present study describes a new approach for appraising rabies virus neutralizing antibodies based upon flow cytometry. This method have been used for detection of a range of different viruses (see McSHARRY, 1994, for review) and we have recently adapted it for monitoring rabies infection in BHK-21 and C6 cell lines by fixed (PV) or wild rabies virus (WRS) strains (BORDIGNON et al., 2002). In addition to detecting and quantifying virus-infected-cells, antibodies to viruses have also been detected and quantified by flow cytometric analysis, such as to HCMV (McHUGH et al., 1986 and 1988), HSV (SCILLIAN et al., 1989) and HIV (SLIGH et al., 1989). Those authors used microspheres coated with viral antigens to detect specific antibodies in serum samples. In the present study, however, the measurement of VNA was performed indirectly, since their presence was disclosed by inhibition of infection.
Since rabies virus nucleocapsid structures are intracytoplasmic, permeabilization of the cells at the time of fixation was necessary to allow antibody conjugate enter the cells. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid. In order to evaluate the humoral immune response against rabies virus in vaccinated humans or animals, the WHO Expert Committee on Rabies recommends that only neutralizing antibodies be measured, since this kind of antibody has extensively been shown to be protective (WIKTOR et al., 1973; DIETZSCHOLD et al., 1987). For that reason, in all tests available for detecting VNA, dilutions of heat-inactivated serum are previously incubated with a fixed amount of rabies virus before being inoculated in a susceptible cell-culture or experimental animals (WEBSTER & DAWSON, 1935; WIKTOR & CLARCK, 1973; SMITH et al., 1973; ZALAN et al., 1979). The neutralizing power of the serum is then evaluated by residual virus infectivity observed on those systems. The most widely used cell-culture techniques are those based on observation of reduction of infected-cell foci by direct fluorescence method (WIKTOR & CLARCK, 1973; SMITH et al. 1973; ZALAN et al., 1979), and the technique described by SMITH et al. (1973) is a reference world-wide. In most of those assays the serum-neutralization end-point titer is defined as the dilution factor that neutralizes 50% to 100% of the challenge inoculum. The titers of antibody in the test serum samples (IU/ml) are obtained by comparison with the titer of a reference standard which must be included in each test. In all tests, the reading are made by optical reading in fluorescence microscopes which turns the work highly arduous and rises the chance of errors when a great number of serum samples are routinely processed.
The flow cytometric method for VNA titration here described uses an analytic automatized procedure for reading. After constructing a standard curve of virus infection inhibition, based on the ability of the reference serum in neutralizing rabies virus, values of any serum test may be obtained by extrapolation. The procedure showed to be very reproducible, as it can be observed by the small standard deviation values from four independent experiments showed in Fig. 1. When the results of VNA titration of 104 dog serum samples obtained by FC were compared to those obtained by SFIMT and ELISA, the values showed a positive correlation (p < 0.01) with r values of 0.7 and 0.6, respectively. As it can be seen in Fig. 2, although FC and SFIMT had a positive correlation, a complete concordance between the methods was not possible. The disagreement was more evident with serum samples that exhibit lower VNA titers in SFIMT, which otherwise could indicate a higher sensitivity of FC. Nevertheless, no specific tests for determining specificity and sensitivity of FC was carried out. For that, it would be necessary a number of negative and positive serum samples. Since the serum samples were obtained from dogs which attend a Regional Vaccination Campaign, we did not know the exact immunization status of the animals. LYNG (1994) had also reported observations about occasional unexplainable differences between relative potencies of a serum obtained by two unrelated methods.
The lower correlation between FC and ELISA (Fig. 2) was easily explainable since the latter technique detect not only neutralizing antibodies but also anti-RNP antibodies.
The study here presented has shown evidences that Flow Cytometry may be used for titration of rabies VNA. Our results permit to envisage the FC as an automated and sensitive technique to evaluate VNA in sera not only from immunized animals but most likely in human serum samples too.
Detecção e quantificação de anticorpos anti-rábicos neutralizantes pela técnica de citometria de fluxo
A titulação de anticorpos neutralizantes contra o vírus rábico (AcN) pós-imunização é um parâmetro aceito como indicador de eficácia da vacina e do sucesso do processo de imunização. Este estudo descreve um teste de neutralização realizado em cultura de células, analisado através da técnica de citometria de fluxo. Diluições seriadas de amostras de soro foram misturadas in vitro com vírus rábico e adicionados a células BHK-21. Após incubação de 24 h, as células foram individualizadas por tratamento com tripsina, fixadas e permeabilizadas com p-formaldeído e coradas com conjugado específico. A porcentagem de inibição da infecção viral causada pelos anticorpos específicos presentes nas amostras de soro foi determinada em citômetro de fluxo Beckton & Dickinson FACSCaliburâ e comparada com um soro de referência internacional. Os títulos de AcN foram determinados por extrapolação. Os níveis de correlação (r) entre os títulos obtidos com CF e SFIMT foi de 0.7712 (p < 0,001), e entre CF e ELISA de 0,6702 (p < 0,001). Os resultados indicam que a CF pode ser utilizada na análise de títulos de anticorpos neutralizantes em amostras de soro, com a vantagem de ser um método automatizado. No entanto, devem ser realizados novos estudos, comparando a CF com as técnicas consideradas como padrão ouro, para que os valores reais de Sensibilidade e Especificidade sejam determinados.
Carlos Roberto Zanetti and Juliano Bordignon are scientific fellowship holders of the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq. We are thankful to the HEMOSC manager for the disposition of the Beckton & Dickinson Flow Cytometer use.
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Received: 22 October 2001
Accepted: 10 May 2002
(1) Departament of Microbiology, Immunology and Parasitology of the Federal University of Santa Catarina (MIP/CCB/UFSC), Campus Universitário da Trindade, 88040-900 Florianópolis, SC, Brazil.
(2) Center of Hemotherapy of Santa Catarina (HEMOSC), Avenida Prof. Othon Gama D'eça, Florianópolis, SC, Brazil.
(3) Pasteur Institute, Avenida Paulista 393, 01311-000 São Paulo, SP, Brazil.
Correspondence to: C.R. Zanetti, Departament of Microbiology, Immunology and Parasitology, Universidade Federal de Santa Catarina. MIP/CCB/UFSC, Campus Universitário da Trindade, 88040-900 Florianópolis, SC, Brazil. Phone: 55 48 331-5206. Fax: 55 48 331-9258. Email: firstname.lastname@example.org