Antigen of Bovine Cutaneous Papilloma Detected by Fluorescent Antibodies*

Lois KITZE SMITItIES AND CARL OLSON

(Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin)

SUMMARY Specific antigen was detected in the bovine cutaneous papil loma by fluorescent-

labeled ant iserum prepared against an infectious extract of bovine papilloma. Antigen was present in the nuclei of cells in the keratohyaline and kcratinized layers of the hyperplast ic cpitheliun1 but not in the germinal basal cells. These findings are similar to those reported for the Shope rabbi t papilloma and the hunlan cutaneous papilloma.

The fluorescent ant ibody method of Coons (8) has been useful in determining the intracellular localization of several animal tumor viruses. With the Rous sarcoma (10) and the polyonla (4) viruses in cell cultures, the sites of viral propagation have been established, and information regarding the de- velopmental events leading to production of ma- ture virus has been obtained with this method. Also, recent studies of the Shope rabbit papi l loma and Rous chicken sarcoma in vivo (6) have demon- s t rated the characteristic distribution of specific viral antigens in the tumor cells of the natura l host.

In the following studies, the fluorescent anti- body method was employed in a search for specific viral antigens in the bovine papilloma.

M A T E R I A L S AND M E T H O D S

Antigen.--Extracts of bovine papillomas con- taining infectious virus, as established by their abili ty to produce papillomas on the skin of test calves, were used as antigens. The extracts were prepared from a 15-~0 per cent suspension of ground bovine warts in 0.15 i NaC1. This suspen- sion was first centrifuged at 480 X g for 20 min- utes in the cold to remove the coarse material, and then at 18,400 )< g for 30 minutes. The sediment was discarded, and the supernatant fluid, which

* Published with the approval of the Director of the Wis- consin Agricultural Experiment Station. Supported in part with the aid of RG, C46~27 National Institutes of tIcalth. The technical assistance of Miss Margaret K. Wilson and the help of Drs. I). F. Brobst and A. J. Luedke in collecting papilloma tissues arc gratefully acknowledged.

Received for tmblication June 80, 1961.

had an infectivity t i ter of 0.1 X 10 -5 to 10 -6 intra- dernlally on the skin of calves, comprised the anti- gen. I t was stored in small aliquots at --65 ~ C. until used either for the immunizat ion of calves or in complement-fixation tests.

Antibody.--Antisera were prepared by injection of the bovine war t extracts into calves. Two calves, 1 ~ months oht and recovered from experi- ment.ally induced papillomatosis, were immunized by three intramuscular injections, 10 days apart , of 10 ml. of wart antigen emulsified with an equal volume of ad juvant (9 nil. Bayol F and 1 ml. Arlaccl A). Sera collected 14 days following the last injection were used to prepare fluorescein con- jugates. These sera had ant ibody titcrs of 1:1~8 determined in a complement-fxat ion test employ- ing bovine papilloma antigen. Whether or not the conlplenlcnt-fixing ant ibody is the one responsible for specific fluorescence, it appeared to be useful in selecting sera suitable for labeling with the dye: conjugates prepared from two normal sera lacking complement-fixing antibodies produced no fluores- cence in sections of bovine papillomas, whereas conjugates made from post- imnmnization sera containing this ant ibody caused specific fluo- rescence in similar sections of the papillomas.

Preparation of labeled globulin.--Ganlnla-globu- lin was precipitated from the sera with 50 per cent sa turated ammonium sulfate. After dialysis against 0.01 M phosphate-buffered saline, pH 7.~, the ~-globulin fractions were freeze-dried, then stored in a desiccator jar at 4 ~ C. Conjugation of 7-globulin with fluorescein isothiocyanate (Syl- vana) was carried out according to a method pre- viously described (5). The final reaction mixture

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1558 Cancer Research Vol. ~1, D e c e m b e r 1961

contained ~00 mg. of freeze-dried 7-globulin, 10 rag. of the dye, 18 ml. of buffered saline (0.01 M phosphate, 0.15 M NaC1, pH 7.~), and ~ ml. of 0.5 ~I carbonate-bicarbonate buffer, pH 9.0. The conjugate was dialyzed at 4 ~ C. for several days against phosphate-buffered saline, pH 7.~, to re- move unconjugated dye and then stored in small aliquots at - 6 5 ~ C. Immedia te ly before use, con- jugates were twice absorbed with acetone-extract- ed bovine liver powder (100 rag. for each ml. of conjugate) (~). The liver powder was prepared from a pool of equal portions of liver taken from eight normal adul t cows, with a view toward re- moving any intra-species tissue ant ibody tha t may have developed in the sera during immunization with the bovine wart preparations.

Staining procedure.--Bovine papillomas were produced by the intradermal injection of bovine wart material into calves. Ninety to 1~0 days after infection, the cauliflower-like growths, approxi- mate ly e-6 inches in diameter, were removed, and representative pieces were stored at - 6 5 ~ C. Frozen sections of the bovine papillomas were cut, air-dried, and used either the same day or within a few days, in which instance they were kept in a desiccator in the refrigerator. Immedia te ly before being stained the sections were fixed for 10 min- utes in acetone and then dried for an hour in a 37 ~ C. incubator. Fluorescein conjugates were ap- plied for 80 minutes at 87 ~ C. After being rinsed, the sections were mounted in buffered glycerol consisting of 3 parts of 0.01 M phosphate buffer, pII 8.0, and 7 parts of glycerol.

Frozen sections of several warts were stained directly with fluorescein-labeled conjugates pre- pared from each of the two post-immunization sera and from two normal bovine sera. The "blocking" control to determine inhibition of specific fluores- cence was carried out by applying either the un- labeled antiserum or normal serum prior to the specific conjugate. Additional controls consisted of

frozen sections of normal bovine skin stained with either the normal serum or immune serum con- jugates. In each experiment, unstained sections were examined for autofluorescence. Subsequent to fluorescent observations, papilloma sections were rinsed in buffered saline and stained with hematoxylin and eosin.

Fluorescent microscope.--The ultraviolet light source was an Osram, type HBO-s mercury arc bulb in a Reichert housing and used with a Spen- cer monocular microscope. A Coming U.V. exciter filter, No. 5480, and a Wrat ten K-~ excluding filter were used.

RESULTS

The morphologic structure of bovine papilloma, described elsewhere in detail (1), is i l lustrated in Figure 1. The hyperplast ic epithelium consists of proliferating basal cells, an intermediate prickle- cell layer, the keratohyaline region, and the super- ficial cornified layer. Specific fluorescence was ob- served in nuclei of cells in the outer layers of the papilloma (Figs. ~, 4). When juxtaposed with photographs of the same fields (Figs. 1, 3) taken after the sections had been counter-stained with hematoxylin and eosin, the antigens of bovine papilloma virus were seen to be confined largely to nuclei in the keratohyaline and keratinized layers; occasionally, a fluorescent nucleus occurred in the deeper prickle cells, but was never seen in the proliferating basal cells or in the cells of the under- lying connective tissue. No specific fluorescence was noted in the cytoplasm of any cell. The green nuclear fluorescence was observed in papilloma sections stained with conjugated anfisera but not with conjugated normal sera and was completely inhibited by a prior application of unlabeled im- mune serum but not by unlabeled normal serum. The epithelium of normal bovine skin showed no fuorescence after being stained with either im- mune or normal serum conjugates absorbed with

FIG. 1.--A section of bovine papilloma stained with hema- toxylin and eosin subsequent to staining with fluorescein-con- jugated antiserum. The field is the same as the fluorescent field shown in Figure ~ aud illustrates the morphologic structure of the papilloma. The hyperplastic epithelium is comprised of proliferating basal cells (A), keratohyaline region (B), and keratinized layer (C). Note correspondence of arrows to those in Figure ~. Mag. X 100.

Fro. ~.--The same field in the section of bovine papilloma shown in Figure 1 but stained with fluorescein-labeled anti- serum. The discrete areas of bright fluorescence are confined to the keratohyaline and keratinized layers of the epithelium. The fuorescent nuclei indicated by arrows occur in the keratinizing region of the prickle-cell layer. Mag. X100.

Fro. 3.--A section of bovine papilloma stained with hema-

toxylin and eosin following the fluorescent observations shown in Figure 4. Note the characteristic epithelial layers in the papilloma. Mag. X100.

Fro. 4.--A fluorescence photomicrograph of the field of bovine papilloma illustrated in Figure 8. Fluorescing antigen is evident against the background autoftuorescence of the eorni- fled layer. Mag. X 100.

FIG. 5.--Fluorescence photomicrograptl of a single nucleus, greatly enlarged, with the characteristic "mot t led" appearance resulting from the irregular distribution of bovine papilloma antigen. The nucleus was in a cell in the keratohyaline region of the papilloma. Mag. X3000.

FIG. 6.--Fluorescence photomicrograph illustrating the as- sociation of bovine papilloma antigens with nuclei and nuclear fragments in the eornified layer of the papilloma. Mag. )<450.

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SMITHIES & OLSON--Bovine Papilloma Antigen and Fluorescent Antibodies 1559

bovine liver powder. In specifically stained sections of papilloma

some vividly fluorescing nuclei were seen in the large, vacuolated cells in the outer region of the prickle-cell layer (Fig. ~). Characteristically, the intensity of the fluorescence varied within the same nucleus, giving it a mottled appearance (Fig. 5). Antigens of bovine papilloma virus were often associated with the flattened, elongated nuclei and nuclear fragments in the cornified layer (Fig. 6). Autofluorescence in bovine papilloma sections was minimal and limited to the superficial, keratinized epithelial layer where it appears (Figs. ~, 4) as a uniform, grayish background fluorescence.

DISCUSSION The characteristic distribution of bovine papil-

loma virus in the outer layers of the tumor is es- sentially similar to the findings in rabbit papilloma (7). Studies of the human wart virus with the elec- tron microscope indicate that this virus, too, is present in the superficial, epithelial layers (8, 11). The similarity of these findings suggests a common pattern of viral development in the cutaneous papillomas of the three species. Noyes and Mellors (1957) postulated from their fluorescent antibody studies of rabbit papilloma that virus, which should be present in the germinal, basal cells to stimulate the characteristic epithelial hyperplasia of the wart, was there, but in some nonantigenic, incomplete form (possibly as viral nucleic acid) and, therefore, not detectable by fluorescent anti- body. This hypothesis would seem to apply as well to the bovine papilloma.

The bovine papilloma, however, differs some- what from the rabbit papilloma in that the con- nective tissue element is usually greater in the bovine wart. Further, under some conditions, a bovine tumor may be produced in which connec- tive tissue comprises the larger part of the growth (1). This fibromatous type of growth is also char- acteristic of the tumor produced in the horse by bovine papilloma virus (9). The intracellular lo-

calization of bovine papilloma virus in tumors con- sisting predominantly of connective tissue is not yet known. In the present study no antigen of bovine papilloma virus was seen in the connective tissue cells of the bovine papillomas examined.

REFERENCES

1. BAGDONAS, V., and OLSON, C. Observations on Immunity in Cutaneous Bovine Papillomatosis. Am. J. Vet. Re- search, 15:~40-45, 1954.

~. COONS, A. H. Fluorescent Antibody Methods. In: J. F. DANIELLI (ed.), General Cytochemical Methods, pp. 399- 4o~. New York: Academic Press, Inc., 1958.

3. COONS, A. H., and KAPLAN, M. H. Localization of Antigen in Tissue Cells. II. Improvements in a Method for the Detection of Antigen by Means of Fluorescent Antibody. J. Exp. Med., 91:1-13, 1950.

4. HENLE, G.; DEINgARDT, F.; and RODHIQ~:F~Z, J. The De- velopment of Polyoma Virus in Mouse Embryo Cells as Revealed by Fluorescent Antibody Staining. Virology, 8: 388-91, 1959.

5. MARSHALL, J. D.; EVELAND, W. C.; and SMITH, C. W. Superiority of Fluorescein Isothiocyanate (Riggs) for Fluorescent Antibody Technic with a Modification of Its Application. Proc. Soc. Exp. Biol. & Med., 98: 898-900, 1958.

6. MELLORS, R. C. Tumor Cell Localization of the Antigens of the Shope Papilloma Virus and the Rous Sarcoma Virus. Cancer Research, 20: 744-46, 1960.

7. MELNICK, J. L. ; BVNTXNa, H. ; BANFIELD, W. G. ; STRAUSS, M. J.; and GAYLORD, W. H. Electron Microscopy of Viruses of Human Papilloma, Molluscum Contagiosum, and Vaccinia, Including Observations on the Formation of Virus within the Cell. Ann. N.Y. Acad. Sci., 54:1~14-~5, 195~.

8. NOYES, W. F., and MELLORS, R. C. Fluorescent Antibody Detection of the Antigens of the Shope Papilloma Virus in Papillomas of the Wild and Domestic Rabbit. J. Exp. Med., 106: 555-6~, 1957.

9. OLSON, C., and COOK, R. H. Cutaneous Sarcoma-like Lesions of the Horse Caused by the Agent of Bovine Papil- loma. Proc. Soc. Exp. Biol. & Med., 77:~81-84, 1951.

10. VOGRT, P. K., and RUBIN, H. Localization of Infectious Virus and Viral Antigens in Chick Fibroblasts during Suc- cessive Stages of Infection with Rous Sarcoma Virus. Virology, 13: 5~8-44, 1961.

11. WILLIAMS, M. G.; HOWATSON, A. F.; and ALMEIDA, J. D. ~[orphologlcal Characterization of the Virus of the Human Common Wart (Verruca Vulgaris). Nature, 189:895-97, 1961.

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1961;21:1557-1559. Cancer Res   Lois Kitze Smithies and Carl Olson  Fluorescent AntibodiesAntigen of Bovine Cutaneous Papilloma Detected by

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