Comparison of three bioassays for rat interferon

Journal of C’irological Methods,

163

9 (1984) 163-171

Elsevier JVM 00333

COMPARISON

GARY

OF THREE BIOASSAYS

R. BURLESON’

and DAVID

FOR RAT INTERFERON

P. HERZOG

Lobund Laboratory, Department of Microbiology, University of Notre Dame, Notre Dame, IN 46556, U.S.A. (Accepted

24 May 1984)

Compared

to the well-characterized

rat interferons. method,

In the present

a hemagglutination

effect. These methods tions to determine systems. features rat

the antiviral,

required.

interferon

bioassay

few studies have been conducted

rat interferon

and a method

and immunoregulatory

desirable

the reduction

bioassay

method,

interferons,

three

characteristics;

influenza

sensitivity,

virus

reduction

role of interferon

effect bioassay

precision,

in viral cytopathic investiga-

in existing rat model

depends

combines

convenience,

on

a plaque-reduction

and thus facilitate

the choice of bioassay

in viral cytopathic

including

bioassays:

involving

in order to better assay rat interferons

antitumor,

has certain Overall,

of a rat interferon

we compared

yield-reduction

were evaluated

Each method

application

murine and human

study,

on the specific

most of the desired

rapidity,

and economy.

hemagglutination

INTRODUCTION

In comparison to the more thoroughly studied human and murine interferon% the literature contains few reports directly pertaining to the characterization and purification of rat interferons (Schonne, 1966; Lin and Abreu, 1979; Schellekens et al., 1980; Poindron et al., 1981). The lack of a good rat interferon bioassay makes it difficult to study the role and effect of interferons in the many excellent rat model systems, including the well-defined tumor models. Various methods to quantify the antiviral activity of interferons have been developed and reviewed (Grossberg et al., 1984). In the present study, three interferon bioassay methods were applied to the rat system: plaque-reduction, hemagglutination yield-reduction (HYR), and reduction in viral cytopathic effect (CPE). These methods were compared rons.

to determine

the bioassay

most appropriate

IRequests for reprints should be addressed ro: Dr. G.R. Burleson, Northrop

Services,

Inc. - Environmental

Sciences,

Health

for studies of rat interfe-

Effects

P.O. Box 12313, Research

Research

Triangle

U.S.A. 2Presenr address: Micromedic

0166-0934/X4/$03.00

Systems,

Inc., 102 Witmer

Road,

0 1984 Elsevier Science Publishers

B.V.

Horsham,

Laboratory,

Park, NC 27709,

PA 19044, U.S.A.

164

MATERIALS

AND METHODS

Cells A fibroblast

cell line derived

from Sprague-Dawley

rat embryos

and routinely

passaged in our laboratory was used in all experiments. This cell line, designated SD, was derived from germ-free Sprague-Dawley rat embryos by Dr. Nehama Sharon at the Lobund Laboratory, University of Notre Dame (Braslawsky, 1974). Subcutaneous injection of lo6 of these cells into the para-lumbar region of weanling SpragueDawley rats does not cause tumor formation (Burleson et al., 1978). The cells used in this study were between passage number 117 and 145. Cells were grown in Eagle’s minimum essential medium with Earle’s salts supplemented with L-glutamine and nonessential amino acids (E-MEM) containing 10% (v/v) heat-inactivated calf serum (E-MEM-CS 10%) (Grand Island Biological Co., Grand Island, NY). Viruses

The recombinant influenza virus, X7(Fl), used in the HYR bioassay was obtained from Dr. S.E. Grossberg at the Medical College of Wisconsin. Kilbourne and coworkers (1967) derived this virus from a backcross between the recombinant influenza virus strain X7 and the A2/R1/5+/57 strain of influenza virus. The X7(Fl) virus was previously used for interferon bioassays utilizing its neuraminidase activity as a measure of virus replication (Sedmak and Grossberg, 1973a,b; Sedmak et al., 1975; Sedmak and Grossberg, 1981). In the present studies, X7(Fl) was passaged in lo-day-old chicken embryos and stored at -70°C as allantoic fluid suspensions. Vesicular stomatitis virus (VSV), Indiana strain, obtained from Dr. M. Pollard at the University of Notre Dame and used in the plaque-reduction and CPE bioassays, was passaged in BHK-21 cells and stored at -7O’C. Interferon

standard

Since an international laboratory rat interferon

reference standard

standard is not available for rat interferon, a was used in every bioassay. This standard was

obtained by intravenous injection of poly I : poly C (400 ug) in Lobund Wistar rats. Rats were bled 2 h after injection and the pooled sera were aliquoted and stored at -20°C. All results were expressed as laboratory units of interferon relative to our laboratory rat interferon standard. The laboratory rat interferon standard was used for all studies on the comparison of rat interferon bioassays. RESULTS

Plaque-reduction

bioassay

The plaque-reduction bioassay for rat interferon was based on the method developed for chicken interferon by Wagner (1961). One milliliter of E-MEM-CS 10%

165

containing

5 X lo5 SD cells was pipetted

into each well of 12-well tissue culture plates

(2.4 X 1.9 cm) (Linbro Scientific, Inc., Hamden, CT). Plates were incubated at 37°C for 3 days. Confluent cell monolayers were then washed twice with 0.15 M NaCl in 0.01 M phosphate buffer pH 7.3 (PBS). Interferon samples were diluted in E-MEM containing 2% calf serum (E-MEM-CS 2%) and 1 ml of this dilution was added to each well of the tissue culture plates. The SD cells and diluted interferon samples were incubated for 24 h. The interferon sample dilutions were then removed, the cell monolayers were washed twice with PBS, and 0.1 ml of VSV was added per well. Vesicular stomatitis virus was diluted in GLB (gelatin, 5 g/l and lactalbumin hydrolysate, 2.5 g/l in Hanks’ balanced salt solution) containing sodium bicarbonate and supplemented with antibiotics. Cells were challenged with the VSV dilution that results in the development of 50 plaques/cell monolayer. Virus was adsorbed at 37°C for 30 min, excess virus was removed, and the cell monolayers in each well were covered with 0.5 ml of an agar overlay. The overlay was composed of 5 parts E-MEM; 1 part tryptose phosphate broth (Difco Laboratories, Inc., Detroit, MI); 3 parts melted algar (Difco Laboratories) in Hanks’ balanced salt solution (30 g/l); and 1 part calf serum. After the overlay hardened, the plates were inverted and incubated for 48 h; 0.5 ml of stain overlay was then added to each well. The stain overlay was prepared like the initial overlay with the addition of 4 ml of 5% (w/v) neutral red/100 ml of overlay. The stain overlay was allowed to harden in the dark; the plates were then inverted and incubated in the dark at 37°C for 4 to 10 h. Plaques were then counted and recorded. The interferon titer for a sample was calculated (by plotting on semilogarithmic paper the logarithm of the reciprocal of the interferon dilution versus the percent of virus control) to determine the plaque depressing dose 50 (PDDS,). The laboratory rat interferon standard had a mean log,, interferon titer of 3.900 when bioassayed by the plaque-reduction method (Table 6). Hemagglutination yield-reduction bioassay The HYR bioassay for rat interferon was a modification of the original bioassay of mouse, human, and chicken interferon using Sindbis or GD VII as challenge virus (Oie et al., 1972). In our bioassay, we used the X7(Fl) recombinant influenza virus (SD cells were resistant to challenge with the hemagglutinating GD VII and encephalomyocarditis viruses, unpubl. results). Initial attempts to infect SD cells with this virus resulted in low hemagglutinin yields. We therefore varied several parameters including cell number, growth conditions, and challenge virus dose. Optimum viral hemagglutinin yields occurred in SD cells in E-MEM-CS 10% containing 0.025 M N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) adjusted to pH 7.5 with 2 N NaOH. One-hundred microliters of SD cells (5 X lo4 cells) were dispensed into each well of 96-well flat bottom plates (Linbro Scientific, Inc., or Falcon, Oxnard, CA). The cells were incubated at 37°C for 24 h and then washed twice with PBS. Interferon samples were diluted in E-MEM-CS 2% + TES using four-fold dilutions

166

and added to the wells (100 ul/well) containing SD cell monolayers. The plates were again incubated for 24 h and the interferon sample dilutions were removed with two PBS washes. Fifty microliters of a 1 : 5 dilution of stock X7(Fl) virus in GLB (multiplicity of infection of 25) was added to each well. The virus was adsorbed at 37°C for 1 h and the cell monolayers were then washed three times with PBS. Cells were incubated in 100 ul of E-MEM-CS 2% + TES at 37°C for an additional 24 h. Cells were then frozen and thawed three times and virus hemagglutinin assayed (Oie et al., 1972; Sedmak and Grossberg+ 1973b). In order to determine optimum conditions for the X7(Fl) hemagglutination (HA) assay, we varied the type of buffer used to dilute virus and red blood cells (RBCs). Doubling dilutions of the virus were prepared in 0.15 M NaCl (saline) with the following buffers: PBS, pH 7.3; TES, pH 6.0; TES, pH 7.0; TES, pH 7.5; and TES, pH 8.0. All buffers contained 0.1% (w/v) bovine serum albumin (BSA). These virus dilutions were prepared (50 ul/well) in 96-well round bottom plates (Linbro Scientific). Human Type 0 RBC suspensions (0.5% v/v) were also prepared in these same buffers and were added to the corresponding wells (50 ul/well). The RBC suspension (10%) was standardized to contain 8 X lOa RBC/ml. Plates were maintained at 4°C during the hemagglutination process. The highest X7(Fl) HA titers were obtained using either saline alone or in TES pH 7.0 as diluting buffer for virus and RBCs (Table 1). At higher pH values, several of the lower dilutions gave apparently negative (no HA) results. It appeared that under these conditions the matrix formed by viral agglutination of RBCs collapsed, producing an effect similar to prozoning. In a second experiment, the RBC concentration and the BSA content of the buffer were varied. Doubling dilutions of the virus were prepared with saline in TES at pH 7.0 or pH 7.5. Each buffer contained 0.1% or 0.5% (w/v) BSA. Dilutions (50 ul/well) were prepared in 96-well round bottom plates. A 0.25% or 0.50% (v/v) suspension of

TABLE

1

Effect of buffer on hemagglutination Buffer

by X7(Fl)

Apparent Reciprocal

Saline

virus

virus HA yield Reciprocal

of last

of first

positive

positive

dilution

dilution

128

8

PBS. pH 7.3

64

8

Saline + TES, pH 6.0

64

4

Saline + TES, pH 7.0

128

4

Saline + TES, pH 7.5

64

8

Saline + TES, pH 8.0

64

16

167

human

Type

maintained

0

RBC in TES

at 4°C during

in the most sensitive settling

was added

hemagglutination.

detection;

time of the pattern.

however,

to each

well (50 l.d/weIl).

Plates

were

Low RBC levels in the HA assay resulted this approach

A 0.5% RBC concentration

was limited by the clarity and resulted

in optimum

patterns

and sensitivity (Table 2). As shown in Table 2, BSA concentrations of 0.1% and 0.5% in the buffer had little effeci on titer. The BSA content did dramatically affect the amount of prozoning observed. Increased BSA concentration caused an increase in the observed prozoning. Interferon titers were calculated by plotting the logarithm of the reciprocal of the interferon dilution versus the logarithm of the decrease in hemagglutinin yield. Decrease in hemagglutinin yield was determined by the number of positive wells observed in the HA assay. The interferon titer is the reciprocal of the dilution that results in a 0.5 loop10 decrease in hemagglutinin yield (Oie et al., 1972). The laboratory rat interferon standard had a mean log,,, interferon titer of 4.364 when bioassayed by the HYR method (Table 6).

The last bioassay evaluated for measurement of rat interferon activity was a reduction in CPE method reported for human interferon (Rubinstein et al., 1981). Several experiments were conducted to determine optimum bioassay conditions. First, the relationship between SD cell density and VSVchallenge dose was examined. Two-fold dilutions of the rat interferon standard were prepared in E-MEM-CS 10% f TES in 96-well flat bottom plates (100 $/well). Then, 100 l.tl of E-MEM-CS 10% -t TES containing 1 X IO4 or 5 X IO4 SD cells/well were added to the interferon dilutions.

TABLE

2

Effect of BSA and RBC concentration Buffer and RBC

on hema~~lutinatjon

Apparent

virus HA yield

0.1%

BSA

Saline, pH 7.0, 0.5% RBC

128

4b

Saline, pH 7.0, 0.25% RBC

2.56

Saline, pH 7.5, 0.5% RBC Saline, pH 7.5, 0.25% RBC

by X7(Fl)

virus

concentrationa

a All saline solutions

BSA

64

32

2

256

32

64

8

64

32

256

2

128

32

buffered

’ First value is the reciprocal dilution.

0.5%

with TES. of the last positive dilution;

second value is the reciprocal

of the first positive

Cells were incubated

with interferon

diluted in E-MEM-CS

at 37°C for 6 h. Vesicular

10% + TES to 1,000 or 5,000 pfu/50

stomatitis

virus was

~1 and was then added to

the wells (50 pi/well) still containing interferon. Plates were incubated at 37°C and the ceils were observed periodically for CPE. When CPE in virus control wells (wells that contained

no interferon

sample)

was complete,

cell monolayers

were washed

and

stained with 0.5% (w/v) crystal violet in 70% (v/v) methanol. (Monolayers were stained 22 to 28 h after the addition of virus.) Interferon titers were determined to be the reciprocal of the interferon dilution that afforded 50% protection (by visual inspection) to the experimental ceil monolayers as compared to virus controls. As had been reported by Rubinstein et al. (1981), lower cell numbers resulted in a loss of interferon sensitivity. A higher ceil density (5 X lo4 SD ceils/well) resulted in a more sensitive interferon bioassay than a lower cell density (1 X lo4 SD ceils/well) (Table 3). The effect of VSV challenge dose was also examined. Rat interferon dilutions were prepared as above. One-hundred microliters of E-MEM-CS 10% -t TES containing 5 X IO4 SD cells was added to each well of 96well flat bottom plates. Plates were incubated at 37’C for 6 h. Each well then received 50 pi of E-MEM-CS 10% -t TES containing 1,000; 5,000; 10,000; or 20,000 pfu/weii of VSV. The CPE bioassay was performed as above. Interferon titers were inversely related to the amount of virus added (Table 4). We also evaluated the effect of time of VSV challenge on the CPE bioassay sensitivity. Rubinstein et al. (1981) report that although some sensitivity is lost, cells and challenge virus can be simultaneously added to dilutions of interferon samples. To evaluate this protocol in the rat interferon system, 1,000 pfu of VSV was added to suspensions of 5 X IO4 SD cells in various dilutions of rat interferon standard at various times after the addition of ceils. A signi~cant increase in sensitivity was observed when the cells and interferon sample were allowed to incubate 6 h prior to VSV addition (Table 5). Comparisons of the precision

TABLE

and sensitivity

of ail three bioassays

were performed

3

Effect of cell density

and VSV challenge

SD cells/well

Interferon

dose on sensitivity

of the CPE rat interferon

bioassay

titer

Virus chalienge (pfu/well) 1.000 1 x 104

1,600”

5 x 104 a Reciprocal monolayer

25,600 of the highest (compared

5,000 400 12,800

dilution

of rat interferon

to virus controls).

standard

that resulted

in 50% protection

of the cell

169

TABLE

4

Effect of VSV challenge

dose on sensitivity

Virus

Titer of

(pfu/well)

interferon

of CPE rat interferon

bioassaya

standardb 1,000

25,600

5,000

12,800

10,000

12,800

20,000

6,400

a SD cell number b Reciprocal monolayer

TABLE

was 5 X lo4 cells/well,

of the highest (compared

dilution

standard

that resulted

in 50% protection

of the cell

5

Effect of time of VSV challenge Time of

on sensitivity

of the CPE rat interferon

bioassay

Titer of

virus

interferon

challenge

(h)

standardb

0

400

3

1,600

6

25,600

a SD cells and rat interferon ’ Reciprocal

of the highest

monolayer

TABLE

of rat interferon

to virus controls).

(compared

combined dilution

at time zero; VSV added

of rat interferon

standard

immediately,

that resulted

3 h, or 6 h later.

in 50% protection

to virus controls).

6

Comparative

precision

and sensitivity

Bioassaya

of three bioassays

Runsb

for rat interferon Mean

Replicates

SD

interferon titer Plaque-reduction

5

5

3.9ooc

0.186’

Hemagglutination

13

19

4.364

0.281

I5

23

4.247

0.210

yield-reduction Cytopathic

effect

inhibition ” Bioassays

performed

under optimum

b Each run was performed ’ Values expressed

on a different

as log,, of interferon

conditions

as stated

in Results.

day. titer as determined

by each bioassay

of the cell

170

under the optimum bioassay conditions determined by the above experiments (Table 6). For the HYR bioassay, X7(Fl) and RBC dilutions were prepared in the following buffer: saline, 0.1% (w/v) BSA, and TES, pH 7.0. For the CPE bioassay, 5 X lo4 SD cells and 1,000 pfu/well of VSV were used. The plaque-reduction bioassay was performed as previously are shown in Table 6.

stated.

Comparisons

of the three bioassays

of rat interferon

DISCUSSION

There is little information in the literature on rat interferon bioassays. The precision of the three rat interferon bioassays compared in this study is comparable to bioassays for interferons of other species (Grossberg et al., 1974). A sigmoidal-type dose response was obtained for the laboratory rat interferon standard with all three bioassay methods. The plaque-reduction bioassay exhibited the least sensitivity of the three, but appeared to have equal or better precision. Inherent disadvantages in the plaque-reduction bioassay included the large volume of cells and media required, the low number of bioassays that could be run concurrently, and the high chance of contamination due to the increased number of manipulations. The HYR and CPE bioassays have comparable sensitivities. The sensitivity of the CPE bioassay can be increased by sacrificing speed (e.g., allowing the cells to incubate with the interferon samples for longer times). Comparison of our results to those of investigators using other rat interferons must await the availability of an international rat interferon reference standard. Based on our observations, the CPE bioassay is the method of choice, especially when large numbers of interferon samples need to be bioassayed. This bioassay can be performed with greater speed and requires fewer manipulations than the plaquereduction or HYR bioassays. The CPE method provides good sensitivity and precision as well as convenience, rapidity, and economy. The CPE method does, however, have drawbacks that preclude abandonment ofthe other bioassays. In the CPE bioassay, the interferon sample is in simultaneous direct contact with the SD cells and the VSV challenge. This arrangement does not preclude a direct inhibiting effect on viral replication by some substance in the sample. Such a substance has been described (Baron and McKerlie, 1981; Hughes et al., 198 1) in some cell cultures. The many common characteristics between our rat laboratory interferon standard and other interferons as well as the activity in the plaque-reduction and HYR bioassays indicate that these inhibiting substances are not the source of activity for the laboratory interferon standard used in this study. Another possible drawback of the CPE bioassay is the apparent difference between the rate of induction of the antiviral state by Type I and Type II interferons. The slower development of the antiviral state in cells treated with Type II interferon (Dianzani et al., 1978) has also been observed in our system. The difference in the rate of induction of the antiviral state can result in different bioassay sensitivities for

171

different

interferon

is employed.

types when a short period of cell contact

We chose a time for incubating

virus addition that afforded a compromise potential problem, convenience, and sensitivity. seem prudent

to evaluate

interferon

prior to virus challenge

interferon samples with SD cells prior to this amon, 0 several factors, including As in all interferon

titers between

work, it would

Type I and Type II interferons

qualitatively and within the same type quantitatively only when the samples to be compared are run simultaneously in the same bioassay. Laboratory reference standards for each type of interferon should be included in each bioassay. ACKNOWLEDGEMENT

This research was supported by Miles Laboratories, Inc., the Art Ehrmann Cancer Fund of the Fraternal Order of Eagles, and Impro Products, Inc. The authors thank Dr. J.J. Sedmak for critical review and comments. The authors also appreciate Karla Gunsolus’s excellent help with manuscript preparation. REFERENCES

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