【病毒外文文獻】2005 Assays for the assessment of neutralizing antibody activities against Severe Acute Respiratory Syndrome (SARS) asso

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under a microscope It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE In the current study we established the utility of two additional assays to SARS associated coronavirus CPE cytopathic effect PR plaque virus type 1 MOI Journal of Immunological Methods 301 2005 21 30 Abbreviations SARS Severe Acute Respiratory Syndrome SCV reduction NRS neutral red staining TCID 50 50 tissue culture measure the neutralizing activities against SCV the plaque reduction PR and the neutral red staining NRS assays The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 HIV 1 In this assay the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A 540 The neutralizing antibody titers can be easily determined with either of the two new assays In this report we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV D 2005 Elsevier B V All rights reserved Keywords Severe Acute Respiratory Syndrome SARS Coronavirus Neutralization assay Anti viral antibody Research paper Assays for the assessment of neutralizing antibody activities against Severe Acute Respiratory Syndrome SARS associated coronavirus SCV Shixia Wang Pavlo Sakhatskyy Te Hui W Chou Shan Lu T Laboratory of Nucleic Acid Vaccines Department of Medicine University of Massachusetts Medical School 364 Plantation Street Lazare Research Building Worcester MA 01605 2397 United States Received 11 January 2005 accepted 6 March 2005 Available online 25 April 2005 Abstract Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome SARS or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus SCV However the current assay based on the cytopathic effect CPE which has been frequently cited in literature has several limitations The CPE assay relies on the visual observation on the damage of SCV infected target cells 0022 1759 see doi 10 1016 j jim 2005 0 multiplicity of infection T Correspondin E mail addr infectious dose HIV 1 human immunodeficiency DMEM Dulbecco modified Eagle medium front matter D 2005 Elsevier B V All rights reserved 3 008 g author Tel 1 508 856 6791 fax 1 508 856 6751 ess shan lu umassmed edu S Lu hard to evaluate Therefore it is difficult to come up with a titration curve to measure the strength of a 1 Introduction The severe acute respiratory syndrome SARS associated coronavirus SCV a new member in Coronaviridae caused highly virulent emerging infec tious disease in human population spreading many parts of the world Drosten et al 2003 Fouchier et al 2003 Ksiazek et al 2003 Kuiken et al 2003 Peiris et al 2003 Poutanen et al 2003 SCV can be transmitted rapidly from person to person with an approximately 11 case fatality rate Although the first epidemic had been successfully contained and only very few new cases were reported after fall of 2003 Enserink 2003 Normile 2004a b SARS still remains a threat due to its highly transmittable nature to human populations and the mysterious origin of SCV Arita et al 2003 Chan et al 2003 Inouye 2003 Ratzan 2003 Sampathkumar et al 2003 Tong and Liang 2004 Currently there are no proven antiviral drugs effective for this viral infection Drosten et al 2003 Holmes 2003 Kuiken et al 2003 Normile 2004b Developing effective vaccines against SCV is the most cost effective approach to achieve protection in a large population susceptible to SCV infection It has been reported that high titers of protective antibodies were present in the convalescent sera of SCVinfected patients and the passive transfer of these sera could improve the clinical outcome of SARS Li et al 2003a Pearson 2004 This implies that if a vaccine can elicit robust humoral immunity it will be protective against SCV infection by eliminating or reducing the cell free viral infectivity Chantler and Davies 1987 Burton 1997 Maruyama et al 1999 Roehrig et al 2001 Burton et al 2004 To evaluate the neutralizing antibody activities in serum samples from either SCV infected hosts or those immunized with candidate SCV vaccines it is critical to establish highly reproducible and quantitative in vitro virus neutralization assays Since the discovery of SARS the neutralizing antibodies against SCV infection have been mainly detected by a simple microneutralization assay based on the cytopathic effect CPE of SCV to its target cells Li et al 2003b Buchholz et al 2004 Sui et al 2004 Zeng et al 2004 This method relies on the S Wang et al Journal of Immunolog22 direct observation of damaged target cells from SCV infection under a microscope However the results neutralizing antibody with serially diluted testing antibodies Traditionally various neutralization assays have been developed for many different viruses Plaque reduction assays have been widely used to evaluate the neutralizing antibody responses against viruses that can form plaques in infected cells such as rubella Rhim and Schell 1967 Sato et al 1979 flavivirus Russell and Nisalak 1967 Ibrahim et al 1968 vaccinia Kitamura et al 1973 Newman et al 2003 and measles viruses Orenstein et al 1987 Vazquez Rosales et al 1996 However unpublished data including our own have observed that the plaques formed in SCV infected target cells such as Vero E6 were poor and could not be visualized clearly unless a microscope is used In the current study we have improved the plaque formation in SCV infected monolayer target cells so the plaques could be easily counted for SCV neutralizing antibody analysis The neutralization assay based on the target cell viability has also been widely used to study the neutralizing activities against HIV 1 in which neutral red dye is used to stain the live and healthy cells that are protected from HIV 1 infection Montefiori et al 1998 In HIV 1 studies the MT 2 cells are used as the target cells Montefiori et al 1988 1998 In the current report we demonstrated that the neutral red method can be used for staining of Vero E6 cell which is a main target cell line for SCV infection 2 Materials and methods 2 1 Antibodies New Zealand White rabbit sera specific for differ ent segments of SCV spike protein were produced by can be influenced by the subjective interpretation from the researchers and it is not easy to quantita tively determine the neutralizing activities based on the degree of cytopathic effect While completely protected cells can be easily distinguished from the damaged cells partially protected cell populations are ical Methods 301 2005 21 30 DNA immunization as recently reported Table 1 Wang et al 2005 Rabbits were immunized with Neutralizing CPE b b 2938 49 2561 44 492 95 4436 55 b independent four bi weekly immunizations by a gene gun and the immune sera tested in this study were collected two weeks after the 4th immunization Sera were stored at 70 8C and heat inactivated for 30 minutes at 56 8C before use 2 2 Cells and virus stocks The African green monkey kidney Vero E6 cells American Type Culture Collection Manassas VA were used for both the propagation of SCV and the neutralization assays The cells were cultured in Dulbecco modified Eagle medium DMEM supple mented with 100 units ml of penicillin G 100 mg ml of streptomycin 2 mM L glutamine and 10 heat inactivated fetal bovine serum FBS All cell cultures were maintained in 5 CO 2 at 37 8C The Urbani strain of SCV used in this study was Table 1 Neutralizing antibody titers against SCV Urbani strain Rabbit Sera Spike DNA vaccines Spike protein fragments Pre bleed N A N A RS001 N A N A RS002 S Full length RS003 S1 Amino acids 12 798 RS004 S2 Amino acids 797 1192 RS005 S1 1 Amino acids 12 535 RS006 S1 2 Amino acids 534 798 N A not applicable T The values are the geometric meansFstandard deviation from 4 S Wang et al Journal of Immunolog obtained from the US Center for Diseases Control and Prevention CDC Atlanta GA All the procedures handling the SCVand infected cell cultures were held in a special biosafety level 3 BSL 3 laboratory under protocol approved by Institutional Biosafety Commit tee IBC To produce the SCV stock the virus was propagated in Vero E6 cells at a multiplicity of infection MOI of 0 01 culture medium was collected at day 4 and filtered through a 0 45 micron filter to remove the cell debris The titer of the virus stock was determined in Vero E6 cell monolayer seeded at 96 well microtiter plates Corning NY with 20 000 cells well The virus 50 tissue culture infectious dose TCID 50 was determined by a 4 day endpoint titration assay as described previously Li et al 2003b 2 3 CPE based microneutralization assay CPE based microneutralization assay was con ducted in 96 well microtiter plates as described Li et al 2003b Three fold dilutions of heat inactivated rabbit sera were tested in triplicate wells for the presence of antibodies that neutralized the infectivity of SCV in monolayer of Vero E6 cells 400 TCID 50 of virus in 50 Al well was incubated with 50 Al of rabbit serum or tissue culture medium for 1 hour at 37 8C After the incubation 20 000 Vero E6 cells in 100 Al were added to each well at MOI of 0 02 Results of neutralization were determined by the appearance of CPE which was observed under a microscope on day 4 of infection The neutralizing antibody titer was defined as the reciprocal of the highest serum dilution at which no CPE breakthrough in any of the triplicate testing wells was observed antibody titersT Plaque reduction Neutral Red staining 30 b30 b30 30 b30 b30 F1946 28 2737 5F565 22 4669 16F707 47 F1478 85 596 01F131 71 5486 36F584 52 F279 33 154 21F24 44 878 63F140 56 F2982 28 3608 08F752 20 8845 09F1389 23 30 b30 b30 assays ical Methods 301 2005 21 30 23 2 4 Plaque reduction based neutralization assay The SCV plaque forming units pfu in the virus stock were determined by a plaque assay in 12 well tissue culture plate Vero E6 cells 0 5C210 6 cells per well were seeded onto the 12 well tissue culture plates the day before infection On the next day 50 pfu 16000TCID 50 equivalents ofSCVin100Alwas first incubated with or without serially diluted antibodies in a total volume of 150 Al in 96 well U bottom tissue culture plates for 1 hour at 37 8C Then the mixture was added into Vero E6 cells seeded 12 well plate after removing their culture medium and incubated for 1 hour at 37 8C The plates were washed with DMEM and the monolayer cells were overlaid with 1 ml of for 30 min at room temperature followed by aspiration removal of the formaldehyde solution and twice PBS wash Monolayers were then stained with 300 Alof 0 5 of crystal violet for 5 min at room temperature and the plates were washed twice with water After the plates were air dried plaques were counted The neutralization was calculated as the percentage of the number of plaques reduced with a particular testing serum as compared to the mean of the plaque counts for the three virus control wells without sera in the same assay The neutralizing antibody titer was determined as the highest serum dilution which achieved 50 plaque reduction 2 5 Neutral red staining NRS based neutralization assay The NRS neutralization assay was performed in 96 well flat bottom plates The set up is similar to that of the CPE based neutralization assay Briefly after incubation of 400 TCID 50 of virus with rabbit serum in a total of 100 Al DMEM medium per well for 1 hour at 37 8C Vero E6 cells 20 000 cells in 100 Al was added to each well at MOI of 0 02 On day 5 of infection when more than 70 cells formed CPE in the viral control wells without any sera the culture medium was removed from the testing wells and 100 Al of 10 neutral red Sigma in DMEM medium was added to each well After incubation for 1 hour at 37 8C the neutral red medium was removed and the plates were washed twice with phosphate buffered saline PBS pH 7 2 Then 100 Al of acid alcohol 1 acetic acid in 50 ethanol was added to each well After incubation for 30 minutes at room temperature the absorbance of neutral red stained plates was read at 540 nm A 540 3 Results 3 1 Neutralization of SCVas measured by CPE assay DMEM medium containing 1 w v of methylcellu lose 4 000 CP Sigma MO After 2 day incubation at 37 8C Vero E6 monolayers were washed with PBS and fixed with 250 Al of pre chilled 4 formaldehyde S Wang et al Journal of Immunolog24 The CPE based neutralization assay was the first and the most frequently used neutralization assay in the SARS research Wang et al 2005 However the way of determination on neutralizing antibody titers has been inconsistent and was poorly described in literature This assay relies entirely on the observation of cell morphology to determine the effect of neutralizing antibody As shown in Fig 1a after being infected with SCV for 4 days Vero E6 cells in the microtiter plate started forming visible CPE including dissociated cell patterns The degree of CPE can be scored as less severe b Q Fig 1a right well or more severe b Q Fig 1a center well as compared to the protected monolayer cells in the presence of positive anti S rabbit sera bC0Q Fig 1a left well when observed under a microscope How ever the degree of morphological changes is hard to be quantified and may be subjected to observersT variation This limitation becomes even more problematic when the titers of neutralizing antibodies need to be determined Fig 1b depicts one sample assay in which the hyperimmune positive anti S rabbit serum was serially diluted at 3 fold dilutions CPE was first observed when the sera dilution reached 1 8100 Fig 1b indicated by However the CPE did not appear in all three testing wells at the same time and the level of CPE in each well also varied data not shown In order to give a consistent scoring of the neutralizing antibody responses the titers of positive sera can be determined either by a more complicated scoring system based on how many wells showed CPE at a given sera dilution or simply based on the last sera dilution when none of the triplicate wells has any visible CPE 1 2700 in the example of Fig 1b By using the latter criteria a panel of rabbit sera elicited by DNA vaccines expressing different forms of SCV S antigen were analyzed Table 1 Both the full length S antigen and the N terminal S1 subunit antigen induced excellent neutralizing antibodies against SCV The C terminal S2 subunit also elicited positive albeit low level neutralizing activities as we recently reported Wang et al 2005 When the S1 subunit protein was further divided into two separate domains S1 1 and S1 2 it was clear that the neutralizing epitope was located at the S1 1 region which has the binding domain for SCV receptor ical Methods 301 2005 21 30 ACE 2 Li et al 2003b The S1 2 region did not show any neutralizing activities Table 1 CPE CPE Serum dilution Triplicate wells 1 100 1 300 1 900 a b S Wang et al Journal of Immunolog While CPE assay is useful the assay to assay variation is significant as shown by high standard deviations Table 1 It is likely due to the variation on the observable changes of CPE when a stringent criteria absent of CPE in any testing wells was applied This problem can become more severe when different researchers are involved in the scoring of CPE CPE assay is also less effective in providing more quantitative measurements of the antibody strength such as the concentration of sera that can inhibit 50 or 90 of infection IC50 or IC90 3 2 Neutralization of SCV measured by plaque reduction assay As noted among researchers working with SCV Vero E6 cells can only form very tiny plaques when 1 2700 1 8100 1 24300 1 72900 1 218700 Fig 1 a Sample pictures of cytopathic effect CPE changes in SCV infected left and with various levels of CPE middle and right b A sample Vero E6 cells following serial dilutions of a positive anti S rabbit serum The detectable CPE in all triplicate wells CPE ical Methods 301 2005 21 30 25 conventional plaque assay technique was used Fig 2a It usually requires high concentration of SCVand the resulting plaques are hard to count even under a microscope To improve the plaque formation we discovered that overlay of methylcellulose with proper concentration and molecular weight 4 000 CP was very critical With this improved method clear plaques were formed in wells infected with SCV while the uninfected cell control well remained free of plaques Fig 2b We then performed the neutralization study in 12 well tissue culture plates with this modified plaque reduction assay To validate the plaque reduction based neutralization assay the positive anti S rabbit serum RS003 and negative control rabbit serum RS001 were used A series of rabbit sera in 3 fold dilutions were added and the numbers of plaques in Defined NAb titer Vero E6 cells observed under a microscope 10C2 without C0 CPE based neutralizing assay showing the appearance of CPE in arrow indicates the last serum dilution which did not show any violet plaques neutralization added each well were counted under a light source Fig 2c The number of plaques in each well increased along Fig 2 Plaque assay plates fixed with formaldehyde and stained by crystal the traditional method b SCV infected Vero E6 cells formed clear overlay method was used c A representative plaque reduction based S rabbit serum RS003 or the negative control rabbit serum RS001 were of these two rabbit sera S Wang et al Journal of Immunolog26 with the dilution of anti S rabbit serum while the number of plaques in the negative sera control wells remained unchanged The percentage of plaque reduction in the presence of positive antibodies was calculated by using the formula below plaque reduction plaque counts in testing well C0 plaque counts in cell control plaque counts in viral control C0 plaque counts in cell control C2100 The neutralizing antibody titer in plaque reduction assay can be reported as the reciprocal serum dilution at which 50 of plague reduction was achieved as compared to the viral control wells By using this assay the neutralizing antibody activities of the same set of positive anti S rabbit sera were analyzed Table 1 While the plaque reduction assay demonstrated a similar pattern on the relative levels of neutralizing antibody activities as the CPE assay the neutralizing titers determined by the plaque reduction assay was in general lower than that achieved by CPE assay This suggested that the plaque reduction assay may be less sensitive than the CPE assay in determining the neutralizing activities against SCV infection 3 3 Neutralization of SCV measured by neutral red staining a Poor plaques formation in SCVinfected Vero E6 cells with as transparent dots 0 5 1 mm in size while the methylcellulose assay with the methycellulose method Serially diluted anti to Vero E6 cells incubated with SCV see Table 1 for description ical Methods 301 2005 21 30 The cell viability upon viral infection can also be measured by neutral red staining Previously we have used this technique to evaluate the cell viability for HIV 1 infected target cell MT 2 The neutral red dye can be taken by the healthy MT 2 cells during staining and will not be washed off by PBS The same neutral red staining method can be applied to Vero E6 cells the target cells for SCV infection despite they are adherent epithelial cells and form monolayer attached to the tissue culture plates After staining and washes the healthy Vero E6 cells will hold the neutral red dye but not t