Journal of Virological Methods, 39 (1992) 55-G’ 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
55
VIRMET 01361
A novel, spectrophotometric microneutralization assay for respiratory syncytial virus Kathleen
L. Rubino
Cancer and Infectious Diseases Research,
and Judith
A. Nicholas
Upjohn Laboratories,
(Accepted 24 February
Kalamazoo,
MI (USA)
1992)
Summary
We describe a simple and rapid microneutralization assay for respiratory syncytial virus (RSV) based on the calorimetric quantitation of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a formazan product by the mitochondria of viable cells. Conditions for RSV infectivity were first optimized for sensitivity and reproducibility based on cell density and on RSV concentration as a function of multiplicity of infection (MOI) and time post-infection and the resulting optical densities were shown to be inversely proportional to MOI. For RSV neutralization, dilutions of heatinactivated human plasma were preincubated with RSV and complement prior to infection of cells in microtiter plates. Following MTT dye conversion, 50% RSV neutralization titers were determined by linear regression analysis of the optical density values and endpoints were markedly influenced by MOI. The MTT-based assay was shown to be comparably sensitive to the plaque reduction assay for quantitation of neutralizing antibody, but more readily adaptable to the screening of a large number of samples. Finally, we demonstrated that the MTT microneutralization assay for RSV was useful for quantitation assay of neutralization activity in sera of mice and cotton rats. Microneutralization;
Cytopathology;
Endpoint titer; RSV
Correspondence to: K.L. Rubino, Cancer and Infectious Diseases Research, Upjohn Laboratones, Henrietta St., Kalamazoo, MI 49007, USA.
301
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Introduction Respiratory syncytial virus (RSV), a human paramyxovirus, is the leading cause of severe respiratory illness in infants under two years of age (Chanock et al., 1976; Glezen et al., 1986). No safe and effective vaccine exists for RSV, however, the disease usually lessens in severity upon the third reinfection with the virus (Henderson et al., 1979; Fernald et al., 1983). The immune responses to RSV have been extensively investigated and the induction of neutralizing antibody in response to either of the two surface glycoproteins, F or G, is associated ‘with the development of host protection (Elango et al., 1986; Olmsted et al., 1986; Stott et al., 1986; Walsh et al., 1987; Wertz et al., 1987). Several methodologies have been described for the detection of RSV neutralizing antibody that are based on pretreatment of virus with dilutions of monoclonal or serum antibody followed by infection of cell monolayers. Residual RSV infectivity has been assayed by either a plaque reduction assay (Coates et al., 1966; Kennedy et al., 1988; Murphy et al., 1989) reduction in RSV antigen expression (Routledge et al., 1988; Anderson et al., 1985; Anderson et al., 1988; Toms et al., 1980), or by a decrease in RSV-induced viral cytopathology (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). Each of these techniques offers adequate detection of RSV neutralizing antibody for a variety of experimental designs, however none is particularly suited for the rapid screening and direct quantitation of a large number of samples. Given the limitations of the existing neutralization assays for RSV, we sought to develop an additional methodology which could be useful for screening large numbers of samples for RSV neutralizing antibody without compromising assay sensitivity. We have developed a rapid, sensitive microneutralization assay in which RSV infectivity is measured after treatment of RSV infected cells with the tetrazolium bromide salt, MTT. MTT is cleaved by dehydrogenase enzyme reduction in the mitochondria of viable cells (Slater et al., 1963; Mossman, 1983) and produces a blue crystal formazan product which, upon solubilization, can be read spectrophotometrically. In this report, we describe the optimization of the MTT assay for reproducible detection of RSV neutralization activity in human, mouse, and cotton rat plasma or sera. To facilitate the software program was quantitation of neutralization titers, a computer developed to determine 50% neutralization endpoints by linear regression analysis. Finally, we demonstrated that the MTT microneutralization assay was comparably sensitive to the standard plaque reduction assay.
Materials and Methods Cells and virus
CV- 1 cells and Hep-2 cells were cultured in Eagle’s minimal essential medium
(EMEM, Whittaker Bioproducts) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Whittaker Bioproducts), 1 mM Hepes buffer (Biologos, Inc.), 0.19% NaHC03 (J.T. Baker) and 50 ,ug/ ml gentamicin sulfate (Whittaker Bioproducts), hereafter referred to as EMEM + 10% FBS. The Long strain of RSV was grown in Hep-2 cells and titered by plaque assay under 0.5% agarose containing EMEM, supplemented as for cell stocks, except with 3% FBS. Recombinant vaccinia viruses, expressing the chimeric FG glycoprotein of RSV (Vat FG), or the gp50 protein of pseudorabies virus (Vat gp50), were as described previously (Nicholas et al., 1991; Marchioli et al., 1990). Plasma and sera
Plasma was obtained from healthy adults (RHE, PBL 40, or PBL 90-3) or an adult convalescent from a diagnosed RSV infection (RSC 001). Sera from cotton rats immunized with alum-precipitated formalin-inactivated RSV or with alum-precipitated FG glycoprotein and sera from cotton rats immunized with alum alone, (Brideau et al., 1989) were generous gifts of Dr. Michael Wathen. Mouse serum samples were collected from untreated BALB/c mice or from BALB/c mice two to three weeks following intranasal infection with lo6 plaque-forming units (pfu) of the Long strain of RSV or two to three weeks following intraperitoneal immunization with lo6 pfu of Vat FG or Vat gp50 as described (Nicholas et al., 1991). Before titration for neutralization activity, all plasma and sera were heat inactivated at 56°C for 30 min to remove any residual complement activity. RSV infection of CV-I cells and MTT
dye conversion
Freshly confluent CV-1 cells were trypsinized and plated onto microtiter plates (Corning) at the indicated cell densities in 50 ~1 volumes in EMEM + 10% FBS. RSV was diluted in the same medium and added in 50 ~1 volumes to the plated CV-1 cells followed by incubation for the indicated number of hours at 37°C. As a control for 0% cell viability, 50 ,~l 3 M guanidine hydrochloride (Sigma) was added in place of medium or virus; uninfected cells were used as a control for 100% cell viability. The MTT assay was performed as follows. Onetenth volume (10 &well) of MTT stock solution (2 mg/ml in PBS, Sigma) was added to each well and plates were incubated for 4 h at 37°C. Culture supernatant fluids were aspirated from the wells, and 100 pi/well of 0.04 N HCl prepared in isopropanol was added for solubilization of the formazan product. Plates were agitated for 2 min to ensure complete solubilization and the optical densities were read at 570 nm on an EIA microplate reader (Bio-tek Instruments, Inc., Burlington, VT). The cytopathic effect (CPE) at each of the MO1 was assessed by comparison of the optical densities of the infected wells to that of the uninfected wells.
58
Neutralization
assays
For neutralization, 60 ~1 of RSV, diluted for the desired final MOI, was preincubated with 30 ~1 of two-fold dilutions of heat inactivated plasma and 30 ~1 low-tox rabbit complement (final dilution, 1:50 in the preincubation mix, Cedarlane Laboratories). All dilutions were performed in EMEM + 10% FBS in triplicate wells of microtiter plates and RSV was the final addition to these preincubation mixtures. For virus infectivity controls, RSV was preincubated with complement and medium, but without plasma or serum. Preincubation mixtures where held on ice for 90 min. CV-1 cells were plated at 2 x lo4 cells/ well in 50 ~1 volumes and cells were then infected with an equal volume of the appropriate preincubation mixture. The MTT assay was performed 72 h later and plates were read as described above. For each plasma tested, virus neutralization titers were determined by comparison of optical densities at each plasma dilution with that of the RSV infectivity control without plasma at the same MOI. The 50% neutralization titer was defined as that plasma dilution which neutralized 50% of the RSV infection, and was then determined by linear regression analysis described below. Plaque reduction assay
RSV neutralization titers in plasma were also determined by testing each plasma’s ability to reduce the number of infectious particles by the standard plaque assay (Coates et al., 1966). Plasma was diluted two-fold in 150 ~1 vol and an equal volume of complement was added (final concentration = 1:50 in the preincubation mix), followed by the addition of 300 ~1 RSV (5 x lo3 pfu/ ml). As controls, preincubation mixtures of RSV with and without complement and without plasma were also prepared. All dilutions were performed in EMEM + 5% FBS and preincubations were held on ice for 90 min. 0.2 ml vol of each mix was used for infection of duplicate 60 mm wells of Hep-2 cells plated the day previously (at 5 x 105/well). Cells were infected at 37°C for 90 min, with occasional agitation, to distribute the inoculum, and cells were overlaid with 0.5% agarose in EMEM + 3% FBS. After 6 days incubation, cells were stained with neutral red (1:33 dilution of 10 mg/ml stock in saline) and plaques were counted. The neutralization titer of a plasma was defined as the greatest dilution which reduced the number of plaques by 50% from the RSV + complement controls without plasma, as calculated by the Reed and Meunch method (Reed and Meunch, 1938). Determination
of RSV neutralization
titers
To automate the RSV MTT microneutralization assay for the determination of both 50% and 0% neutralization endpoint titers, an IBM PC program written in RSjl was developed (BBN Software Products Corp.). The optical densities associated with uninfected cell controls, with cells infected at a range
59
of MOI, and with cells in microneutralization assays were read with an ELISA microplate reader (Biotek) and transferred to a floppy disk. An RSV standard control curve was generated for each experiment, by plotting the optical density against the RSV dilution (MOI) and the MO1 intersection, which was the optical density at the specific MO1 used for the neutralization assays, was determined. A curve was generated for each plasma or serum neutralization by plotting the optical density against the antibody dilution and by using a least squares linear fit. A straight line was generated using the linear portion of the sigmoidal curve and the point at which this line intersected the MO1 intersection represented the 0% neutralization titer. From these data, the 50% neutralization titer was calculated.
Results Effects of RSV infection on MTT
dye conversion
To establish a reproducible and sensitive system for detection of RSV infectivity, we determined the effects of varying cell density, MO1 and assay incubation times on MTT dye conversion. CV-I cells were plated at three different cell densities with eight different MO1 of RSV, MTT dye conversion was quantitated at 24 h, 48 h or 72 h post-infection, and these optical ‘densities were compared to those from uninfected controls. At 24 h post-infection, only a slight change in optical density was observed between uninfected and infected cells at any cell or virus concentration tested (data not shown). Optical densities from both 48 h and 72 h post-infection demonstrated that MTT dye conversion was directly proportional to cell density (Fig. 1, compare at MO1 = 0) and indirectly proportional to the MO1 (Fig. 1). The slope of the curves generated by plotting optical density vs. increasing MO1 increased from 24 h to 48 h (not shown) and from 48 h to 72 h (Fig. 1). At 72 h post-infection a MO1 of 1.25 or greater produced the maximal decrease in MTT dye conversion (Fig. 1). From these data we demonstrated that infection of 2 x IO4 CV-1 cells with RSV at a MO1 of 1.25 for 72 h provided the optimum conditions for detection of virus infectivity on which to base the RSV neutralization assay. Reproducibility
of neutralization
of RSV by human plasma
Next, we determined the variability of the MTT dye conversion assay for measurement of RSV specific neutralizing activity. We obtained human plasma from an adult human donor, RSC 001, convalescent from RSV infection diagnosed by viral isolation a few months prior to plasma donation. To determine interassay variability, we titrated the neutralization activity of aliquots of this plasma stored at 4°C in seven independent assays over a period of several weeks. As shown in Fig. 2, MTT dye conversion was reproducible in both uninfected CV-1 cultures and CV-1 cells infected with RSV following
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0
.08
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31
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77.5 2.5
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50
100
/
MOI Fig. 1. Effect of RSV infection on MTT dye conversion. CV-I cells were plated into microtiter wells at 1 x IO4(A), 2 x lo4 (a), or 4 x lo4 (0) cells/well and were infected with two-fold dilutions of RSV or cells plated at each density were mock-infected with medium for uninfected controls. At 48 h or 72 h postinfection cultures were treated with MTT for dye conversion, as described in the Materials and Methods, and the optical densities (O.D.) were read at 570 nm. Cells treated with 3 M guanidine hydrochloride had O.D. values < 0.05 and represented 0% cell viability. Data are mean O.D. of triplicate wells following infection and MTT dye conversion.
preincubation with medium and complement alone at a MO1 of 1.25 (RSV control cells). RX 001 plasma neutralized RSV infectivity and this neutralization was titrated out with higher plasma dilutions. We observed minimal assay variation between neutralization profiles of each experiment and a direct relationship between the human plasma concentration and optical density. Also included in these assays, but not shown, were controls for RSV infectivity at a range of MO1 for additional monitoring of infection in the absence of human plasma. These data suggested that the optical density observed following preincubation of RSV with human plasma was directly proportional to the neutralizing activity of the plasma. From Fig. 2 we estimated the 50% endpoint to be approximately 2000. Additionally, the 50% neutralization titer, calculated as a mean from each of the seven independent assays, was 2200, which closely approximated the value obtained from the composite shown in Fig. 2.
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Plasma
Dilution100(log2)
Fig. 2. Reproducibility of neutralization of RSV infection by human plasma. Two-fold dilutions of human plasma RSC 001 were preincubated with complement and RSV for a final MO1 of 1.25and plated with CV-1 cells (a). At 72 h post-infection, cultures were treated with MTT for dye conversion, as described in the Materials and Methods. All data given in Fig. 2 are the mean O.D. and standard errors from seven individual experiments and data points are given for both the uninfected controls (0) and the RSV infectivity controls for an MO1 of 1.25(A).
RSC 001
0
1
2
3
4
Plasma
5
6
7
Dilution
8
9
10
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13
1 OO(log2)
Fig., 3. Effect of varying MO1 on RSV neutralization. Plasma RSC 001 was tested in MTT RSV neutralization assays performed at MO1 of 5.0 (a), 1.25 (O), or 0.63 (A) as described in the Materials and Methods. Data were analyzed as described and the 50% RSV neutralization titers are given in the Results section.
Effect of varying MOI on neutralization
endpoints
To further characterize the MTT microneutralization assay for RSV we determined the effect of varying the MO1 on the determination of the 50% neutralization endpoints of three adult plasma having a wide range of RSV neutralization activities (50% neutralization titers from 1540 to 121, as shown in Table 1). For each serum, neutralization assays were performed at MOI’s of 5.0, 1.25, and 0.63 and the 50% endpoint titers were determined by computer
62 TABLE 1 Comparison of the MTT assay for measurement plaque reduction assay Plasma
_ RX
001
RHE
PBL 40
Dilution
of RSV-neutralization
Plaque/well
activity in plasma with the
50% neutralization
titer
Plaque reductiona
MTTb
_
195
_
_
200 400 800 1600 3200 6400
3 5 11 44 104 116
2990
1540
40 80 160 320 640 1280
10
167
117
ZZ 129 139 131
40 80 160 320 640 1280
17 41 70 155 163 178
201
121
“50% neutralization titers were determined by plaque reduction assay calculated according to the Reid-Muench method (Reid and Muench, 1938). b50% neutralization titers determined by MTT microneutralization assay were calculated by linear regression analysis.
analysis. Fig. 3 shows the effect of assay MO1 on the neutralization profiles of plasma RSC 001 and is representative of those profiles obtained with the other two plasmas. An increase or decrease in MO1 resulted in a corresponding increase or decrease in the slope of the neutralization curve for all three plasma samples. Furthermore, decreasing the MO1 resulted in an increased neutralization titer, whereas increasing the MO1 resulted in a decreased neutralization titer. The 50% neutralization titers determined at increasing MO1 for plasma RSC 001 were 1348, 1219, and 512; for plasma RHE were 157, 117, and 57; for plasma PBL 40 were 213, 186, and 67. Ther.efore, these data suggested that the neutralization profiles and endpoint titers of each of these plasma were highly dependent upon the RSV MO1 of the assay. Comparison assay
of the MTT microneutralization
assay with the plaque reduction
It was important to compare the sensitivity of the MTT microneutralization assay to other standard assays used to measure RSV neutralizing antibody. We
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0
4b
0.
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Serum Dilution lO(log *)
Fig. 4. (a) RSV neutralization activity in RSV or FG immunized cotton rats. Cotton rat sera samples were prepared and assayed for RSV neutralization activity, as described in the Materials and Methods, with a starting antibody dilution of 1:40. Values given are mean O.D. of triplicate wells for uninfected CV-I cells (m), RSV infected CV-1 cultures without antibody (O), or cultures infected with RSV plus dilutions of cotton rat sera from animals immunized with: alum (0 - -O), formalin-inactivated RSV (A- - -A) or 200 ng of FG glycoprotein (a- - -0). (b) RSV neutralization activity in RSV infected BALB/c mice compared to naive mice. BALB/c mice were intranasally infected with RSV, as described in Materials and Methods, and after two to three weeks, sera samples were collected and assayed for RSV neutralization activity, along with sera from untreated mice. Optical density values are means of triplicate wells for RSV treated mice (-) or naive mice (-).
selected the plaque reduction assay and compared the 50% endpoint titers of this assay calculated by the method of Reed and Meunch (Reed and Meunch, 1938) with the 50% endpoint titers obtained by the MTT microneutralization assay calculated by linear regression computer analysis. The 50% endpoint titration comparisons are shown in Table 1. For each of the three plasma, the MTT microneutralization assay appeared to be comparably sensitive to the plaque reduction assay on determination of the 50% neutralization endpoints. RSV neutralization
activity in cotton rat and mouse sera
Because it is difficult to find human sera definitely void of neutralizing antibody to RSV, owing to either active or passive (maternal) immunity, and because mice and cotton rats are useful animal models for studying immunity to RSV, we designed the following experiments to compare data collected from
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negative control (naive) sera to data obtained from animals specifically immunized with RSV antigens. Fig. 4a compares the neutralization profile of serum collected from a cotton rat immunized with alum alone (control serum) to that of sera obtained from cotton rats immunized either with formalininactivated RSV or with the chimeric FG glycoprotein prepared as an alum precipitate. The highest tested concentration of control serum (1:40) produced a slight increase in optical density from the virus infectivity control and was suggestive of virus neutralization. Extensive viral CPE was observed, however, through direct microscopic evaluation of these cultures. Additional dilutions of control serum did not neutralize RSV infection, since optical densities of these cultures were about equivalent to the RSV infectivity control (Fig. 4a). In contrast, sera from both RSV- or FG-immunized animals were neutralizing, as indicated by optical densities, which were significantly greater than the RSV infectivity control at high concentrations and the ability to titrate out this activity over several dilutions (Fig. 4a). Further direct microscopic examination of these cultures did not reveal CPE. The 50% neutralization titer induced by RSV immunization was 149, and the 50% titer induced by immunization with FG was 160, as determined by linear regression analysis. Similarly, we assayed and compared RSV neutralization titers from naive BALB/c mice or mice infected intranasally with RSV. Fig. 4b compares the neutralization profiles of immune mouse sera to sera from naive mice. At the most concentrated antibody dilution (1:40) the optical densities for two of the three naive control sera were slightly higher than the RSV infectivity control. However, microscopic evaluation of these cultures revealed extensive viral CPE, suggesting that these higher optical densities associated with high concentrations of naive sera were not evidence of specific neutralization of RSV infection. All sera of RSV-primed mice were neutralizing, as indicated by optical densities, which were significantly greater than the RSV infectivity control, and the ability to titrate out this activity over several dilutions of sera (Fig. 4b). The 50% neutralization titers were 92, 106 and 172 (Fig. 4b). The MTT microneutralization assay was also used for the determination of neutralizing antibody induced by immunization of BALB/c mice, with recombinant vaccinia virus expressing either the RSV chimeric FG glycoprotein (Vat FG) or the gp50 protein of pseudorabies virus (Vat gp50). RSV specific neutralization activity was apparent for pooled sera from Vat FG immunized mice (data not shown) and the mean for the 50% titers determined from four experiments was 146, with about 15% variation between experiments. Additionally, nonspecific neutralization, described previously for high concentrations of serum from naive mice, was apparent from the neutralization profiles obtained from sera from Vat gp50 immunized mice and microscopic evaluation of these wells revealed extensive viral cytopathology. From numerous assays with multiple samples of naive (or non-RSV immune) plasma or sera we estimated the lower limit of assay sensitivity to be 1:40 more than 95% of the time (with a probability of obtaining a higher level of nonspecific neutralization of less than 5%).
Discussion We have described a novel procedure for the quantitation of serum RSV neutralizing activity based upon the calorimetric conversion of MTT into a crystalline formazan product which, following solubilization, can be easily quantitated by an ELISA plate reader. The MTT calorimetric assay has been a useful technology for measuring cytotoxicity, cellular proliferation, or activation in a variety of systems (Mossman, 1983; Ferrari et al., 1990; Heeg et al., 1985; Pearlman et al., 1988; Plumb et al., 1989). In original studies, Mossman demonstrated that the intensity of the formazan product generated upon MTT treatment of mouse lymphoma cells was directly proportional to the number of viable cells and their state of activation and that the dye conversion occurred in the mitochondria of viable cells (Mossman, 1983). Our data confirm and extend these initial studies by demonstrating that the MTT assay can also be used to measure viral infectivity and neutralization of viral infectivity based on an increase in optical density over the virus infectivity controls. Our studies demonstrated that the quantitation of neutralizing activity in plasma or serum was interdependent upon cell density, MO1 and assay incubation times. This observation is consistent with the notion of accumulation of virus-induced cytopathic effects on the viability and mitochondrial activity of infected cells resulting from virus replication and shut-down of host macromolecular synthesis. Thus, the MO1 selected for the MTT neutralization studies was shown to significantly influence the 50% neutralization titers. Our microneutralization assay offers several advantages over other methodologies, which are used to assay RSV neutralizing activity (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). The MTT assay has a relatively short incubation time, the MTT dye conversion reaction itself is simple, requires no washing procedures and results can be quantitated spectrophotometrically and analyzed quickly by either visual estimation or by linear regression analysis. In addition to the ease of the MTT assay, results were shown to be highly reproducible. The MTT assay was shown to be comparably sensitive to the RSV plaque reduction assay for measurement of virus neutralizing antibody. However, we found that high concentrations of naive or negative control serum produced slight to modest increases in optical density compared to the RSV infectivity control, suggestive of neutralizing activity. This problem could be overcome by microscopic examination of these cultures, similar to the methodology used by others (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). Microscopic evaluation revealed extensive viral cytopathology and thus confirmed the non-specific interference with virus infectivity at high serum concentrations using the MTT dye conversion endpoint. Therefore, we have defined the lower limit of our assay at an antibody dilution of 1:40, although additional ‘tine-tuning’ of the MTT assay may be possible for elimination this
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‘non-specific activity’ observed at high serum concentrations. In contrast, the MTT neutralization assay had no apparent upper limits of sensitivity, since we reproducibly determined neutralizing titers in samples having a very wide range of neutralizing activities. These studies describe an additional neutralization assay for RSV, which is easily adaptable for the screening of a large number of samples over an extensive series of dilutions in back-to-back comparisons. We have successfully used this assay to screen at least 140 human plasma samples having 50% neutralization titers ranging from 50 to 1295 (our unpublished data). Although we have not extensively compared our MTT assay to all other methodologies, data presented herein prove our MTT assay for RSV to be a useful tool in investigations of the humoral responses to RSV.
Acknowledgement The authors wish to thank Norman D. Young for his diligent assistance the development of the linear regression analysis computer program.
in
References Anderson, L.J., Hierholzer, J.C., Bingham, P.G. and Stone, Y.O. (1985) Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J. Clin. Microbial. 22, 1050-1052. Anderson, L.J., Bingham, P. and Hierholzer, J.C. (1988) Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J. Viral. 62, 42324238. Beeler, J.A. and van Wyke Coelingh, K. (1989) Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function. J. Virol. 63, 2941-2950. Brideau, R.J., Walters, R.R., Stier, M.A. and Wathen, M.W. (1989) Protection of cotton rats against human respiratory syncytial virus by vaccination with a novel chimeric FG glycoprotein. J. Gen. Virol. 70, 2637-2644. Chanock, R.M., Kim, H.W., Brandt, C.D. and Parrott, R.H. (1976) Respiratory syncytial virus. In: A.S. Evans (ed), Viral Infections of Humans. Plenum, NY, pp. 3655382. Coates, H.W., Alling, D.W. and Chanock, R.M. (1966) An antigenic analysis of respiratory syncytial virus isolation by a plaque reduction neutralization test. Am. J. Epidemiol. 83, 299313. Elango, N., Prince, G.A., Murphy, B.R., Venkatesan, S., Chanock. R.M. and Moss, B.R. (1986) Resistance to human respiratory syncytial virus infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein. Proc. Natl. Acad. Sci. USA 83, 19061910. Fernald, G.W., Almond, J.R. and Henderson, F.W. (1983) Cellular and humoral immunity in recurrent respiratory syncytial virus infections. Pediatr. Res. 17, 753-758. Ferrari, M., Fornasiero, M.C. and Isetta, A.M. (1990) MTT calorimetric assay for testing cytotoxic activity in vitro. J. Immunol. Methods 131, 1655172. Glezen, W.P., Taber, L.H., Frank, A.L. and Kasel, J.A. (1986) Risk of primary infection and reinfection with respiratory syncytial virus. Am. J. Dis. Child 140, 543-546. Heeg, K., Reimann, J., Kabelitz, D., Hardt, C. and Wagner, H. (1985) A rapid calorimetric assay for the determination of IL-2 producing helper T cell frequencies. J. Immunol. Methods 77,237246.
67 Henderson, F.W., Collier, A.M., Clyde, W.A., Jr. and Denny, F.W. (1979) Respiratory syncytial virus infections, reinfections, and immunity. N. Engl. J. Med. 300, 53&534. Kennedy, H.E., Jones, B.V., Tucker, E.M., Ford, N.J., Clarke, S.W., Furze, J., Thomas, L.H. and Stott, E.J. (1988) Production and characterization of bovine monoclonal antibodies to respiratory syncytial virus. J. Gen. Virol. 69, 3023-3032. Marchioli, C.C., Yancey, R.J., Petrovskis, E.A., Timmins, J.G. and Post, L.E. (1987) Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujesky’s disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells. J. Virol. 61, 3977-3982. Mosmann, T. (1983) Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. Murphy, B.R., Sotnikov, A., Paradisio, P.R., Hildreth, S.W., Jenson, A.B., Baggs, R.B., Lawrence, L., Zubak, J.J., Chanock, R.M., Beeler, J.A. and Prince, G.A. (1989) Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease. Vaccine 7, 533-540. Nicholas, J.A., Rubino, K.L., Levely, M.E., Meyer, A.L. and Collins, P.L. (1991) Cytotoxic T cell activity against the 22-kDa protein of human respiratory syncytial virus (RSV) is associated with a significant reduction in pulmonary RSV replication. Virology 182, 664672. Olmsted, R.A., Elango, N., Prince, G.A., Murphy, B.R., Johnson, P.R., Moss, B., Chanock, R.M. and Collins, P.L. (1986) Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual contributions of the F and G glycoproteins to host immunity. Proc. Natl. Acad. Sci. USA 83, 7462-7466. Pearlman, E., Jiwa, A.H., Engleberg, N.C. and Eisenstein, B.I. (1988) Growth of Legionella pneumophzla in a human macrophage-like (U937) cell line. Microbial. Pathogenesis 5, 87-95. Plumb, J.A., Milroy, R. and Kaye, S.B. (1989) Effects of the Ph dependence of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazohum bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 49, 44354440. Reed, L.J. and Muench, H. (1938) A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493497. Routledge, E.G., Willcocks, M.M., Samson, A.C.R., Morgan, L., Scott, R., Anderson, J.J. and Toms, G.L. (1988) The purification of four respiratory syncytial virus proteins and their evaluation as protective agents against experimental infection in BALB/c mice. J. Gen. Viral. 69, 293-303. Slater, T.F., Sawyer, B. and Strauli, U.D. (1963) Studies on succinate-tetrazolium reductase systems III. Points of coupling of four different tetrazohum salts. Biochim. Biophys. Acta 77, 383. Stott, E.J., Ball, L.A., Young, K.K.-Y., Furze, J. and Wertz, G.W. (1986) Human respiratory syncytial virus glycoprotein expressed from a recombinant vaccinia virus vector protects mice against live virus challenge. J. Viral. 60, 607613. Toms, G.L., Gardner, P.S., Pullman, C.R., Scott, M. and Taylor, C. (1980) Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation. J. Med. Virol. 5, 351-360. Trudel, M., Nadon, F., Seguin, C., Dionne, G. and Lacroix, M. (1987) Identification of a synthetic peptide as part of a major neutralization epitope of respiratory syncytial virus. J. Gen. Virol. 68, 2273-2280. Walsh, E.E., Hall, C.B., Briselli, M., Brandriss, M.W. and Schlesinger, J.J. (1987) Immunization with glycoprotein subunits of respiratory syncytial virus to protect cotton rats against viral infection. J. Infect. Dis. 155, 119881204. Wathen, M.W., Brideau, R.J. and Thomsen, D.R. (1989) Immunization of cotton rats with the human respiratory syncytial virus F glycoprotein produced using a baculovirus vector. J. Infect. Dis. 159, 255-264. Wertz, G.W., Stott, E.J., Young, K.K.-Y., Anderson, K. and Ball, L.A. (1987) Expression of the fusion protein of human respiratory syncytial virus from recombinant vaccinia virus vectors and protection of vaccinated mice. J. Virol. 61, 293-301.
55
VIRMET 01361
A novel, spectrophotometric microneutralization assay for respiratory syncytial virus Kathleen
L. Rubino
Cancer and Infectious Diseases Research,
and Judith
A. Nicholas
Upjohn Laboratories,
(Accepted 24 February
Kalamazoo,
MI (USA)
1992)
Summary
We describe a simple and rapid microneutralization assay for respiratory syncytial virus (RSV) based on the calorimetric quantitation of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a formazan product by the mitochondria of viable cells. Conditions for RSV infectivity were first optimized for sensitivity and reproducibility based on cell density and on RSV concentration as a function of multiplicity of infection (MOI) and time post-infection and the resulting optical densities were shown to be inversely proportional to MOI. For RSV neutralization, dilutions of heatinactivated human plasma were preincubated with RSV and complement prior to infection of cells in microtiter plates. Following MTT dye conversion, 50% RSV neutralization titers were determined by linear regression analysis of the optical density values and endpoints were markedly influenced by MOI. The MTT-based assay was shown to be comparably sensitive to the plaque reduction assay for quantitation of neutralizing antibody, but more readily adaptable to the screening of a large number of samples. Finally, we demonstrated that the MTT microneutralization assay for RSV was useful for quantitation assay of neutralization activity in sera of mice and cotton rats. Microneutralization;
Cytopathology;
Endpoint titer; RSV
Correspondence to: K.L. Rubino, Cancer and Infectious Diseases Research, Upjohn Laboratones, Henrietta St., Kalamazoo, MI 49007, USA.
301
56
Introduction Respiratory syncytial virus (RSV), a human paramyxovirus, is the leading cause of severe respiratory illness in infants under two years of age (Chanock et al., 1976; Glezen et al., 1986). No safe and effective vaccine exists for RSV, however, the disease usually lessens in severity upon the third reinfection with the virus (Henderson et al., 1979; Fernald et al., 1983). The immune responses to RSV have been extensively investigated and the induction of neutralizing antibody in response to either of the two surface glycoproteins, F or G, is associated ‘with the development of host protection (Elango et al., 1986; Olmsted et al., 1986; Stott et al., 1986; Walsh et al., 1987; Wertz et al., 1987). Several methodologies have been described for the detection of RSV neutralizing antibody that are based on pretreatment of virus with dilutions of monoclonal or serum antibody followed by infection of cell monolayers. Residual RSV infectivity has been assayed by either a plaque reduction assay (Coates et al., 1966; Kennedy et al., 1988; Murphy et al., 1989) reduction in RSV antigen expression (Routledge et al., 1988; Anderson et al., 1985; Anderson et al., 1988; Toms et al., 1980), or by a decrease in RSV-induced viral cytopathology (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). Each of these techniques offers adequate detection of RSV neutralizing antibody for a variety of experimental designs, however none is particularly suited for the rapid screening and direct quantitation of a large number of samples. Given the limitations of the existing neutralization assays for RSV, we sought to develop an additional methodology which could be useful for screening large numbers of samples for RSV neutralizing antibody without compromising assay sensitivity. We have developed a rapid, sensitive microneutralization assay in which RSV infectivity is measured after treatment of RSV infected cells with the tetrazolium bromide salt, MTT. MTT is cleaved by dehydrogenase enzyme reduction in the mitochondria of viable cells (Slater et al., 1963; Mossman, 1983) and produces a blue crystal formazan product which, upon solubilization, can be read spectrophotometrically. In this report, we describe the optimization of the MTT assay for reproducible detection of RSV neutralization activity in human, mouse, and cotton rat plasma or sera. To facilitate the software program was quantitation of neutralization titers, a computer developed to determine 50% neutralization endpoints by linear regression analysis. Finally, we demonstrated that the MTT microneutralization assay was comparably sensitive to the standard plaque reduction assay.
Materials and Methods Cells and virus
CV- 1 cells and Hep-2 cells were cultured in Eagle’s minimal essential medium
(EMEM, Whittaker Bioproducts) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Whittaker Bioproducts), 1 mM Hepes buffer (Biologos, Inc.), 0.19% NaHC03 (J.T. Baker) and 50 ,ug/ ml gentamicin sulfate (Whittaker Bioproducts), hereafter referred to as EMEM + 10% FBS. The Long strain of RSV was grown in Hep-2 cells and titered by plaque assay under 0.5% agarose containing EMEM, supplemented as for cell stocks, except with 3% FBS. Recombinant vaccinia viruses, expressing the chimeric FG glycoprotein of RSV (Vat FG), or the gp50 protein of pseudorabies virus (Vat gp50), were as described previously (Nicholas et al., 1991; Marchioli et al., 1990). Plasma and sera
Plasma was obtained from healthy adults (RHE, PBL 40, or PBL 90-3) or an adult convalescent from a diagnosed RSV infection (RSC 001). Sera from cotton rats immunized with alum-precipitated formalin-inactivated RSV or with alum-precipitated FG glycoprotein and sera from cotton rats immunized with alum alone, (Brideau et al., 1989) were generous gifts of Dr. Michael Wathen. Mouse serum samples were collected from untreated BALB/c mice or from BALB/c mice two to three weeks following intranasal infection with lo6 plaque-forming units (pfu) of the Long strain of RSV or two to three weeks following intraperitoneal immunization with lo6 pfu of Vat FG or Vat gp50 as described (Nicholas et al., 1991). Before titration for neutralization activity, all plasma and sera were heat inactivated at 56°C for 30 min to remove any residual complement activity. RSV infection of CV-I cells and MTT
dye conversion
Freshly confluent CV-1 cells were trypsinized and plated onto microtiter plates (Corning) at the indicated cell densities in 50 ~1 volumes in EMEM + 10% FBS. RSV was diluted in the same medium and added in 50 ~1 volumes to the plated CV-1 cells followed by incubation for the indicated number of hours at 37°C. As a control for 0% cell viability, 50 ,~l 3 M guanidine hydrochloride (Sigma) was added in place of medium or virus; uninfected cells were used as a control for 100% cell viability. The MTT assay was performed as follows. Onetenth volume (10 &well) of MTT stock solution (2 mg/ml in PBS, Sigma) was added to each well and plates were incubated for 4 h at 37°C. Culture supernatant fluids were aspirated from the wells, and 100 pi/well of 0.04 N HCl prepared in isopropanol was added for solubilization of the formazan product. Plates were agitated for 2 min to ensure complete solubilization and the optical densities were read at 570 nm on an EIA microplate reader (Bio-tek Instruments, Inc., Burlington, VT). The cytopathic effect (CPE) at each of the MO1 was assessed by comparison of the optical densities of the infected wells to that of the uninfected wells.
58
Neutralization
assays
For neutralization, 60 ~1 of RSV, diluted for the desired final MOI, was preincubated with 30 ~1 of two-fold dilutions of heat inactivated plasma and 30 ~1 low-tox rabbit complement (final dilution, 1:50 in the preincubation mix, Cedarlane Laboratories). All dilutions were performed in EMEM + 10% FBS in triplicate wells of microtiter plates and RSV was the final addition to these preincubation mixtures. For virus infectivity controls, RSV was preincubated with complement and medium, but without plasma or serum. Preincubation mixtures where held on ice for 90 min. CV-1 cells were plated at 2 x lo4 cells/ well in 50 ~1 volumes and cells were then infected with an equal volume of the appropriate preincubation mixture. The MTT assay was performed 72 h later and plates were read as described above. For each plasma tested, virus neutralization titers were determined by comparison of optical densities at each plasma dilution with that of the RSV infectivity control without plasma at the same MOI. The 50% neutralization titer was defined as that plasma dilution which neutralized 50% of the RSV infection, and was then determined by linear regression analysis described below. Plaque reduction assay
RSV neutralization titers in plasma were also determined by testing each plasma’s ability to reduce the number of infectious particles by the standard plaque assay (Coates et al., 1966). Plasma was diluted two-fold in 150 ~1 vol and an equal volume of complement was added (final concentration = 1:50 in the preincubation mix), followed by the addition of 300 ~1 RSV (5 x lo3 pfu/ ml). As controls, preincubation mixtures of RSV with and without complement and without plasma were also prepared. All dilutions were performed in EMEM + 5% FBS and preincubations were held on ice for 90 min. 0.2 ml vol of each mix was used for infection of duplicate 60 mm wells of Hep-2 cells plated the day previously (at 5 x 105/well). Cells were infected at 37°C for 90 min, with occasional agitation, to distribute the inoculum, and cells were overlaid with 0.5% agarose in EMEM + 3% FBS. After 6 days incubation, cells were stained with neutral red (1:33 dilution of 10 mg/ml stock in saline) and plaques were counted. The neutralization titer of a plasma was defined as the greatest dilution which reduced the number of plaques by 50% from the RSV + complement controls without plasma, as calculated by the Reed and Meunch method (Reed and Meunch, 1938). Determination
of RSV neutralization
titers
To automate the RSV MTT microneutralization assay for the determination of both 50% and 0% neutralization endpoint titers, an IBM PC program written in RSjl was developed (BBN Software Products Corp.). The optical densities associated with uninfected cell controls, with cells infected at a range
59
of MOI, and with cells in microneutralization assays were read with an ELISA microplate reader (Biotek) and transferred to a floppy disk. An RSV standard control curve was generated for each experiment, by plotting the optical density against the RSV dilution (MOI) and the MO1 intersection, which was the optical density at the specific MO1 used for the neutralization assays, was determined. A curve was generated for each plasma or serum neutralization by plotting the optical density against the antibody dilution and by using a least squares linear fit. A straight line was generated using the linear portion of the sigmoidal curve and the point at which this line intersected the MO1 intersection represented the 0% neutralization titer. From these data, the 50% neutralization titer was calculated.
Results Effects of RSV infection on MTT
dye conversion
To establish a reproducible and sensitive system for detection of RSV infectivity, we determined the effects of varying cell density, MO1 and assay incubation times on MTT dye conversion. CV-I cells were plated at three different cell densities with eight different MO1 of RSV, MTT dye conversion was quantitated at 24 h, 48 h or 72 h post-infection, and these optical ‘densities were compared to those from uninfected controls. At 24 h post-infection, only a slight change in optical density was observed between uninfected and infected cells at any cell or virus concentration tested (data not shown). Optical densities from both 48 h and 72 h post-infection demonstrated that MTT dye conversion was directly proportional to cell density (Fig. 1, compare at MO1 = 0) and indirectly proportional to the MO1 (Fig. 1). The slope of the curves generated by plotting optical density vs. increasing MO1 increased from 24 h to 48 h (not shown) and from 48 h to 72 h (Fig. 1). At 72 h post-infection a MO1 of 1.25 or greater produced the maximal decrease in MTT dye conversion (Fig. 1). From these data we demonstrated that infection of 2 x IO4 CV-1 cells with RSV at a MO1 of 1.25 for 72 h provided the optimum conditions for detection of virus infectivity on which to base the RSV neutralization assay. Reproducibility
of neutralization
of RSV by human plasma
Next, we determined the variability of the MTT dye conversion assay for measurement of RSV specific neutralizing activity. We obtained human plasma from an adult human donor, RSC 001, convalescent from RSV infection diagnosed by viral isolation a few months prior to plasma donation. To determine interassay variability, we titrated the neutralization activity of aliquots of this plasma stored at 4°C in seven independent assays over a period of several weeks. As shown in Fig. 2, MTT dye conversion was reproducible in both uninfected CV-1 cultures and CV-1 cells infected with RSV following
60
4
:
:
:
:
:
0
.08
.15
31
62
:
:
77.5 2.5
:
:
50
100
/
MOI Fig. 1. Effect of RSV infection on MTT dye conversion. CV-I cells were plated into microtiter wells at 1 x IO4(A), 2 x lo4 (a), or 4 x lo4 (0) cells/well and were infected with two-fold dilutions of RSV or cells plated at each density were mock-infected with medium for uninfected controls. At 48 h or 72 h postinfection cultures were treated with MTT for dye conversion, as described in the Materials and Methods, and the optical densities (O.D.) were read at 570 nm. Cells treated with 3 M guanidine hydrochloride had O.D. values < 0.05 and represented 0% cell viability. Data are mean O.D. of triplicate wells following infection and MTT dye conversion.
preincubation with medium and complement alone at a MO1 of 1.25 (RSV control cells). RX 001 plasma neutralized RSV infectivity and this neutralization was titrated out with higher plasma dilutions. We observed minimal assay variation between neutralization profiles of each experiment and a direct relationship between the human plasma concentration and optical density. Also included in these assays, but not shown, were controls for RSV infectivity at a range of MO1 for additional monitoring of infection in the absence of human plasma. These data suggested that the optical density observed following preincubation of RSV with human plasma was directly proportional to the neutralizing activity of the plasma. From Fig. 2 we estimated the 50% endpoint to be approximately 2000. Additionally, the 50% neutralization titer, calculated as a mean from each of the seven independent assays, was 2200, which closely approximated the value obtained from the composite shown in Fig. 2.
61
4 0
:
1
:
:
I
I
:
:
I
:
:
I
:
-1
2
3
4
5
6
7
8
9
10
11
12
13
Plasma
Dilution100(log2)
Fig. 2. Reproducibility of neutralization of RSV infection by human plasma. Two-fold dilutions of human plasma RSC 001 were preincubated with complement and RSV for a final MO1 of 1.25and plated with CV-1 cells (a). At 72 h post-infection, cultures were treated with MTT for dye conversion, as described in the Materials and Methods. All data given in Fig. 2 are the mean O.D. and standard errors from seven individual experiments and data points are given for both the uninfected controls (0) and the RSV infectivity controls for an MO1 of 1.25(A).
RSC 001
0
1
2
3
4
Plasma
5
6
7
Dilution
8
9
10
11
12
13
1 OO(log2)
Fig., 3. Effect of varying MO1 on RSV neutralization. Plasma RSC 001 was tested in MTT RSV neutralization assays performed at MO1 of 5.0 (a), 1.25 (O), or 0.63 (A) as described in the Materials and Methods. Data were analyzed as described and the 50% RSV neutralization titers are given in the Results section.
Effect of varying MOI on neutralization
endpoints
To further characterize the MTT microneutralization assay for RSV we determined the effect of varying the MO1 on the determination of the 50% neutralization endpoints of three adult plasma having a wide range of RSV neutralization activities (50% neutralization titers from 1540 to 121, as shown in Table 1). For each serum, neutralization assays were performed at MOI’s of 5.0, 1.25, and 0.63 and the 50% endpoint titers were determined by computer
62 TABLE 1 Comparison of the MTT assay for measurement plaque reduction assay Plasma
_ RX
001
RHE
PBL 40
Dilution
of RSV-neutralization
Plaque/well
activity in plasma with the
50% neutralization
titer
Plaque reductiona
MTTb
_
195
_
_
200 400 800 1600 3200 6400
3 5 11 44 104 116
2990
1540
40 80 160 320 640 1280
10
167
117
ZZ 129 139 131
40 80 160 320 640 1280
17 41 70 155 163 178
201
121
“50% neutralization titers were determined by plaque reduction assay calculated according to the Reid-Muench method (Reid and Muench, 1938). b50% neutralization titers determined by MTT microneutralization assay were calculated by linear regression analysis.
analysis. Fig. 3 shows the effect of assay MO1 on the neutralization profiles of plasma RSC 001 and is representative of those profiles obtained with the other two plasmas. An increase or decrease in MO1 resulted in a corresponding increase or decrease in the slope of the neutralization curve for all three plasma samples. Furthermore, decreasing the MO1 resulted in an increased neutralization titer, whereas increasing the MO1 resulted in a decreased neutralization titer. The 50% neutralization titers determined at increasing MO1 for plasma RSC 001 were 1348, 1219, and 512; for plasma RHE were 157, 117, and 57; for plasma PBL 40 were 213, 186, and 67. Ther.efore, these data suggested that the neutralization profiles and endpoint titers of each of these plasma were highly dependent upon the RSV MO1 of the assay. Comparison assay
of the MTT microneutralization
assay with the plaque reduction
It was important to compare the sensitivity of the MTT microneutralization assay to other standard assays used to measure RSV neutralizing antibody. We
63 4a
.
0
4b
0.
2
3
4
5
6
7
8
9
10
11
12
13
Serum Dilution lO(log *)
Fig. 4. (a) RSV neutralization activity in RSV or FG immunized cotton rats. Cotton rat sera samples were prepared and assayed for RSV neutralization activity, as described in the Materials and Methods, with a starting antibody dilution of 1:40. Values given are mean O.D. of triplicate wells for uninfected CV-I cells (m), RSV infected CV-1 cultures without antibody (O), or cultures infected with RSV plus dilutions of cotton rat sera from animals immunized with: alum (0 - -O), formalin-inactivated RSV (A- - -A) or 200 ng of FG glycoprotein (a- - -0). (b) RSV neutralization activity in RSV infected BALB/c mice compared to naive mice. BALB/c mice were intranasally infected with RSV, as described in Materials and Methods, and after two to three weeks, sera samples were collected and assayed for RSV neutralization activity, along with sera from untreated mice. Optical density values are means of triplicate wells for RSV treated mice (-) or naive mice (-).
selected the plaque reduction assay and compared the 50% endpoint titers of this assay calculated by the method of Reed and Meunch (Reed and Meunch, 1938) with the 50% endpoint titers obtained by the MTT microneutralization assay calculated by linear regression computer analysis. The 50% endpoint titration comparisons are shown in Table 1. For each of the three plasma, the MTT microneutralization assay appeared to be comparably sensitive to the plaque reduction assay on determination of the 50% neutralization endpoints. RSV neutralization
activity in cotton rat and mouse sera
Because it is difficult to find human sera definitely void of neutralizing antibody to RSV, owing to either active or passive (maternal) immunity, and because mice and cotton rats are useful animal models for studying immunity to RSV, we designed the following experiments to compare data collected from
64
negative control (naive) sera to data obtained from animals specifically immunized with RSV antigens. Fig. 4a compares the neutralization profile of serum collected from a cotton rat immunized with alum alone (control serum) to that of sera obtained from cotton rats immunized either with formalininactivated RSV or with the chimeric FG glycoprotein prepared as an alum precipitate. The highest tested concentration of control serum (1:40) produced a slight increase in optical density from the virus infectivity control and was suggestive of virus neutralization. Extensive viral CPE was observed, however, through direct microscopic evaluation of these cultures. Additional dilutions of control serum did not neutralize RSV infection, since optical densities of these cultures were about equivalent to the RSV infectivity control (Fig. 4a). In contrast, sera from both RSV- or FG-immunized animals were neutralizing, as indicated by optical densities, which were significantly greater than the RSV infectivity control at high concentrations and the ability to titrate out this activity over several dilutions (Fig. 4a). Further direct microscopic examination of these cultures did not reveal CPE. The 50% neutralization titer induced by RSV immunization was 149, and the 50% titer induced by immunization with FG was 160, as determined by linear regression analysis. Similarly, we assayed and compared RSV neutralization titers from naive BALB/c mice or mice infected intranasally with RSV. Fig. 4b compares the neutralization profiles of immune mouse sera to sera from naive mice. At the most concentrated antibody dilution (1:40) the optical densities for two of the three naive control sera were slightly higher than the RSV infectivity control. However, microscopic evaluation of these cultures revealed extensive viral CPE, suggesting that these higher optical densities associated with high concentrations of naive sera were not evidence of specific neutralization of RSV infection. All sera of RSV-primed mice were neutralizing, as indicated by optical densities, which were significantly greater than the RSV infectivity control, and the ability to titrate out this activity over several dilutions of sera (Fig. 4b). The 50% neutralization titers were 92, 106 and 172 (Fig. 4b). The MTT microneutralization assay was also used for the determination of neutralizing antibody induced by immunization of BALB/c mice, with recombinant vaccinia virus expressing either the RSV chimeric FG glycoprotein (Vat FG) or the gp50 protein of pseudorabies virus (Vat gp50). RSV specific neutralization activity was apparent for pooled sera from Vat FG immunized mice (data not shown) and the mean for the 50% titers determined from four experiments was 146, with about 15% variation between experiments. Additionally, nonspecific neutralization, described previously for high concentrations of serum from naive mice, was apparent from the neutralization profiles obtained from sera from Vat gp50 immunized mice and microscopic evaluation of these wells revealed extensive viral cytopathology. From numerous assays with multiple samples of naive (or non-RSV immune) plasma or sera we estimated the lower limit of assay sensitivity to be 1:40 more than 95% of the time (with a probability of obtaining a higher level of nonspecific neutralization of less than 5%).
Discussion We have described a novel procedure for the quantitation of serum RSV neutralizing activity based upon the calorimetric conversion of MTT into a crystalline formazan product which, following solubilization, can be easily quantitated by an ELISA plate reader. The MTT calorimetric assay has been a useful technology for measuring cytotoxicity, cellular proliferation, or activation in a variety of systems (Mossman, 1983; Ferrari et al., 1990; Heeg et al., 1985; Pearlman et al., 1988; Plumb et al., 1989). In original studies, Mossman demonstrated that the intensity of the formazan product generated upon MTT treatment of mouse lymphoma cells was directly proportional to the number of viable cells and their state of activation and that the dye conversion occurred in the mitochondria of viable cells (Mossman, 1983). Our data confirm and extend these initial studies by demonstrating that the MTT assay can also be used to measure viral infectivity and neutralization of viral infectivity based on an increase in optical density over the virus infectivity controls. Our studies demonstrated that the quantitation of neutralizing activity in plasma or serum was interdependent upon cell density, MO1 and assay incubation times. This observation is consistent with the notion of accumulation of virus-induced cytopathic effects on the viability and mitochondrial activity of infected cells resulting from virus replication and shut-down of host macromolecular synthesis. Thus, the MO1 selected for the MTT neutralization studies was shown to significantly influence the 50% neutralization titers. Our microneutralization assay offers several advantages over other methodologies, which are used to assay RSV neutralizing activity (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). The MTT assay has a relatively short incubation time, the MTT dye conversion reaction itself is simple, requires no washing procedures and results can be quantitated spectrophotometrically and analyzed quickly by either visual estimation or by linear regression analysis. In addition to the ease of the MTT assay, results were shown to be highly reproducible. The MTT assay was shown to be comparably sensitive to the RSV plaque reduction assay for measurement of virus neutralizing antibody. However, we found that high concentrations of naive or negative control serum produced slight to modest increases in optical density compared to the RSV infectivity control, suggestive of neutralizing activity. This problem could be overcome by microscopic examination of these cultures, similar to the methodology used by others (Trudel et al., 1987; Wathen et al., 1989; Beeler and van Wyke Coelingh, 1989). Microscopic evaluation revealed extensive viral cytopathology and thus confirmed the non-specific interference with virus infectivity at high serum concentrations using the MTT dye conversion endpoint. Therefore, we have defined the lower limit of our assay at an antibody dilution of 1:40, although additional ‘tine-tuning’ of the MTT assay may be possible for elimination this
66
‘non-specific activity’ observed at high serum concentrations. In contrast, the MTT neutralization assay had no apparent upper limits of sensitivity, since we reproducibly determined neutralizing titers in samples having a very wide range of neutralizing activities. These studies describe an additional neutralization assay for RSV, which is easily adaptable for the screening of a large number of samples over an extensive series of dilutions in back-to-back comparisons. We have successfully used this assay to screen at least 140 human plasma samples having 50% neutralization titers ranging from 50 to 1295 (our unpublished data). Although we have not extensively compared our MTT assay to all other methodologies, data presented herein prove our MTT assay for RSV to be a useful tool in investigations of the humoral responses to RSV.
Acknowledgement The authors wish to thank Norman D. Young for his diligent assistance the development of the linear regression analysis computer program.
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