Clin. exp. Immunol. (1975) 21, 329-338.

AN IMMUNOFLUORESCENCE STUDY OF WARTS P. V. SHIRODARIA AND R. S. MATTHEWS* Department of Microbiology and Immunobiology, The Queen's University of Belfast, and * Department of Dermatology, Royal Victoria Hospital, Belfast (Received 2 January 1975)

SUMMARY

An investigation of the incidence of wart virus-specific antibody and of virion antigens in patients with single or multiple warts taken from different anatomical sites showed that all warts did not contain antigen. The incidence was identical using either rabbit antiserum or human sera known to contain virus-specific antibody. The warts from sole, heel and toe had a much higher incidence of stainable virus antigen. Virus-specific staining was mainly found in the keratinized and granular layers of the wart but occasional synthesis in prickle cell layer was observed. All the patients who had virus antigens in their warts did not have virus-specific antibodies but no patient who had antibody lacked antigen. Of the virus-specific antibodies, the IgM class was predominant; a smaller number also contained virusspecific IgG antibody; none had specific IgG alone. All patients with regressing warts had virus antigens and both classes of virus-specific antibody. A wart cellspecific IgM was also found along with virus-specific antibodies and independent of them. An IgM producing fibrillar staining in human embryonic lung cells was noted in some sera.

INTRODUCTION A preliminary immunofluorescence survey of antibodies to warts and wart virus from groups of patients with regressing or non-regressing warts, suggested that the method of healing or regression may be causally related to antibody responses. In particular, cellular reactions in regression are inconspicuous; anticellular antibody of the IgM class may be present in non-regressing warts and in patients who have no antibody to wart virus; and regressing warts are associated strongly with the presence of wart virus-specific IgG and IgM (Matthews & Shirodaria, 1973). In that survey we suggested that it was necessary to compare the incidence of antibodies and wart virus in the same individuals. The aim of the present survey was to investigate this relationship. In addition a further description of the Correspondence: Dr P. V. Shirodaria, Department of Microbiology and Immunobiology, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BN, Northern Ireland. K

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fluorescent antibody patterns is given with some data on virus antigen in warts taken from different anatomical sites.

MATERIALS AND METHODS

Antigen Non-venereal warts were excised or curetted from the skin under local anaesthetic and stored at - 20'C. They were minced and then ground with sand in distilled water. The suspension was clarified and the supernatant fluid centrifuged at 143,000 g for 1 hr. The pellet was resuspended in phosphate-buffered saline and purified on a sucrose density gradient as described previously (Matthews & Shirodaria, 1973).

Rabbit antisera Rabbit antibody to purified virus was prepared as described in the previous publication (Matthews & Shirodaria, 1973). The rabbits' pre-immune serum and rabbit anti-SV40 serum were used as controls. The sera were absorbed with acetone-treated mouse liver powder and human liver powder at 40C. The titre of rabbit anti-wart virus serum was determined by indirect immunofluorescence. The limiting titre of the serum was found to be 1:64.

Patients' sera Sera were collected from patients suffering from warts. They were stored in small aliquots at - 20'C. Before immunofluorescence tests they were absorbed at 40C with acetone-treated human liver powder. Absorbed sera were inactivated at 560C for 30 min. Sera which showed wart virus-specific IgM were further absorbed with heat-aggregated Cohn Fraction II human gamma globulin (HGG). This was to exclude secondary fluorescent staining of virus antigen due to rheumatoid factor (Shirodaria, Fraser & Stanford, 1973). Wart sections Wart lesions were excised under local anaesthesia, snap-frozen in liquid nitrogen and stored at - 70'C. These biopsies were obtained at the same time as the blood samples. The lesions were sectioned at 5 gum on a Pierce-Slee cryostat. The sections were allowed to dry at room temperature for 20 min, fixed in acetone at room temperature for 10 min and stored at -200C.

Immunofluorescence technique (i) Fluorescein conjugates. Sheep anti-rabbit globulin (Wellcome Reagents Ltd) and sheep anti-human IgG (Wellcome Reagents Ltd) were each conjugated with fluorescein isothiocyanate (FITC) (Sigma Chemical Company) in our laboratory. Sheep anti-human IgM conjugated with FITC (Wellcome Reagents Ltd) was used. The specificity of the conjugate was assessed in indirect viral immunofluorescent systems (Chantler & Haire, 1972). The conjugates were absorbed with acetone-treated mouse liver powder, HEp2 cells and wart-tissue homogenate at 4°C. All the conjugates were used at their optimum staining titres. (ii) Fluorescent staining. For tracing viral antigen, sections from each individual wart were

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331

stained with rabbit sera at a dilution of 1:15 for 1 hr at 37 C and after washing the section sheep anti-rabbit conjugate was applied for 45 min at 37 C. Sections were washed and mounted in buffered glycerol-saline (pH 8 3). For detecting antibody, human sera were applied to wart sections known to contain virus antigen at a dilution of 1:6. followed by appropriate antihuman conjugates. The sera were tested on selected serial positive sections taken from one lesion. The preparations were examined with a Reichart Fluropan microscope and suitable specimens were photographed in a Zeiss standard R.A. fluorescence photomicroscope. (iii) Specificity of staining. The specificity of the rabbit anti-wart virus serum was established by: (a) the absence of staining with pre-immune rabbit serum or with rabbit antiSV40 serum; (b) the loss of staining after the absorption of immune serum with purified wart virus. The specificity of the human antiviral staining was established by the fact that the pattern of positive staining was similar to that obtained with rabbit anti-wart virus serum and also the loss of specific staining after the absorption of positive sera with purified wart virus. RESULTS

Antigen distribution Since the test material for the presence of fluorescent antibody is the wart itself, it was necessary to assess the chances of finding antigen in any section from a single wart and also the likelihood of finding antigen in two or more warts from the same patient. Clinical considerations prevent the examination of many warts from one patient, but the examination of pairs of warts and a few multiple warts taken at the same time from each patient showed that (Table 1) when one wart lesion displayed virus antigen all others did. TABLE 1. Incidence of wart virus antigen in multiple warts

Number of warts from each patient

Total number of patients 14 All positive All negative Different

Pairs

Threes

Fours

Fives

8 4 4 0

4 1 3 1

1

1

1 0

l 0

The reverse was true, a negative wart meant no antigen except in one patient out of fourteen, it was found that out of three wart lesions examined, one showed the presence of the virus antigen in few cells while the other two were negative. It was also observed that when one wart lesion contained a large quantity of antigen, the other lesions from the same patient showed a similar quantity of antigen and vice versa. Furthermore, numerous sections cut from wart lesions revealed that when a wart contained antigen, it was present throughout the lesion. Similarly lesions which contained no antigen were consistently negative throughout the lesion. The sections from antigen-negative warts were further tested with human sera known to contain virus-specific antibody but no antigen was found. The sections from

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antigen-negative warts were also tested with conjugated human anti-IgG and conjugated human anti-IgM globulins to determine whether antigen was present in the form of antigenantibody complexes. The examination of several antigen-negative warts showed that this was not the case. With regard to anatomical site, Cubie (1972) has reported that plantar warts differ from others in respect of virus yield. The distribution of fluorescent staining antigen in warts according to site and age of the lesion is shown in Table 2. Warts in pressure areas have stainable virus significantly more often than other warts. TABLE 2. Incidence of wart virus antigen at different sites in relation to duration of lesion

Site Duration (months) 1-6 7-12 > 12

Total

Finger and hand

Sole, heel and toe

Others

10/18* 6/10 2/14 18/42

35/38t

3/5

4/7+ 0/7 39/52

1/4 4/9

* Number positive/number tested. t Six patients were rejecting their warts. + Two patients were rejecting their warts.

FIG. 1. Cryostat section of wart which shows virion antigens in prickle cell layer. Stained with rabbit anti-wart virus serum and FITC-conjugated anti-rabbit globulin. (Magnification x 455.)

Although antigen-negative and antigen-positive warts were found in all ages, it was found that warts that have been present for less than 1 year tended to have more virus antigen than older warts. There was also great variation in the distribution of the amount of virus antigen between warts of similar age. In some cases it was found largely in the keratin and

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333

granular layers. Some specific staining in the nuclei of the lower region of the granular layer strongly suggested that virus antigen is sited in the nucleoli. Also there was clear evidence of occasional virus synthesis in the prickle cell layer (Fig. 1). It is interesting that in this series of patients, the regressing warts were also found on the sole, heel and toe. Antibody (i) Antibody specificities. Comparative studies of antibodies and of classes of antibody were carried out on serial sections from one wart lesion. However, the results were repeatable on other wart tissue sections. Staining of two different specificities was seen, one, which we shall refer to as virion antigen, being wart virus-specific as determined also by rabbit antiwart virus serum (Fig. 2) and the pattern was found to differ with the class of antibody used. The other was cell-specific confined to the cell membrane of the wart cells. In addition, deposits of immunoglobulin IgM and IgG were observed in areas around the blood vessels in the section.

FIG. 2. Cryostat section of wart showing distribution of virus antigen. Stained with rabbit anti-wart virus serum and FITC-conjugated anti-rabbit globulin. (Magnification x 175.)

FIG. 3. Cryostat section of wart showing fluorescence pattern revealed by human IgM. Stained with a human serum and FITC-conjugated antihuman IgM. (Magnification x 175.)

The 1gM staining of virion antigen was granular (Fig. 3). The granules appeared to be aggregates of virus and were not visible with immunoglobulin IgG staining. IgM staining showed staining throughout the nucleus and ring staining of the nuclear margin. In contrast, IgG staining of virus antigen gave a much smoother appearance (Fig. 4) and nuclear and ring staining was also present. These staining properties of IgM and IgG were both removed from the sera by purified wart virus. The anticellular staining was confined to the IgM class of antibody. The staining of the cell margins (Fig. 5) was frequently obtained by sera which contained virus-specific antibodies, but it was also produced by sera from patients with warts who had no virus-specific antibody. In cases where both virus-specific and anticellular IgM antibody were present, it was difficult to distinguish anticellular staining because of bright virus-specific staining.

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FIG. 4. Cryostat section of wart showing fluorescence pattern revealed by human IgG. Stained with a human serum and FITC-conjugated anti-human

IgG. (Magnification

x

175.)

FIG. 5. Cryostat section of wart showing anticellular IgM staining pattern. Stained with positive human serum and FITC-conjugated anti-human IgM. (Magnification x 175.)

However, when an area of section was selected where there was no virus antigen, the anticellular staining was easily observed. Furthermore, the anticellular staining was distinguishable from virus-specific staining because the absorption of the sera with purified wart virus did not remove the anticellular staining. The anticellular staining was not observed with rabbit sera prepared against highly purified wart virus. In a preliminary experiment ten sera from patients which had both virus-specific and anticellular antibodies and ten sera from patients which had only anticellular antibody were tested on acetone-fixed human embryonic lung fibroblast cultures. The IgM anticellular staining obtained with these sera on tissue culture cells was of a fibrillar type (Fig. 6), similar to that reported by Haire (1972) and it was often very intense. (ii) Relationship of antibody to other features. Two factors will influence an antibody response: (a) the intensity of the stimulus; (b) the duration. The relationship between the length of history of warts and presence of virus-specific IgM and IgG in patients with virus antigen in their lesions is given in Table 3. The correlation between the occurrence of stainable amounts of antigen in the wart and virus-specific antibody in the patient is strong. Thirty-five patients out of fifty-nine had virus-specific IgM antibody and seventeen out of fifty-nine had virus-specific IgG antibody. Although the number of patients who had warts longer than 6 months was small, the proportion showing the presence of antibody cannot be said to be significantly greater than the others. It was interesting to find that some patients who had virus antigen in their warts had neither the virus-specific IgM nor the IgG class of antibody in their sera. The results also show that there was a prolongation of the IgM class of antibody response in patients with persisting wart virus antigen. The patients with regressing warts had virus-specific antibodies of both immunoglobulin

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FIG. 6. Human embryonic lung fibroblasts (tissue culture) showing a fibrillar pattern of staining with positive human serum and FITC-conjugated anti-human IgM. (Magnification x 672.)

classes, confirming our previous observations. Although in patients with non-regressing warts the IgM class of antibody was predominant, a small number of them also had the IgG class of antibody. Table 4 shows the incidence of virus-specific and anticellular antibodies in patients with antigen-positive and antigen-negative lesions at different sites. In the antigen-positive group of patients who had wart lesions at different sites, there was TABLE 3. Duration of infection and virus-specific antibody response in patients with virus antigen

Antibody positive Duration (months) 1-6 7-12 >12

Total

Number tested

Total

46 10 3

59

Antibody negative

IgM

IgG

Total

27

27

14

19

(6)*

(6)

(6)

(0)

6

6

3

4

(2)

(2)

(2)

(0)

2

2

0

1

(0)

(0)

(0)

(0)

35

35

17

24

* Number of patients rejecting their warts.

no significant difference in the virus-specific or the anticellular antibody response. However, the 'other' group was probably too small for comparison. The correlation between the absence of virus-specific antibody and the absence of replicating virus as judged by fluorescent antibody staining was absolute in all thirty-nine cases examined. Yet twenty-two of the thirty-nine patients had antibody which specifically stained wart cells and this anticellular IgM was also the commonest fluorescent staining antibody in the antigen-positive group of

patients.

P. V. Shirodaria and R. S. Matthews

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TABLE 4. Incidence of wart virus-specific and anticellular antibody in patients with antigenpositive and antigen-negative lesions at different sites

Antigen-negative

Antigen-positive Site

Virus-specific

IgM Finger and hand Sole, heel and toe Others

IgG

Anticellular IgM

10/18*

3/18

12/18

24/37t

13/37t

26/37t

1/4

1/4

2/4

Virus-specific

Anticellular

IgM

IgG

IgM

0/24 0/10 0/5

0/24 0/10 0/5

14/24 6/10 2/5

* Number positive/number tested. t Eight patients were rejecting their warts.

DISCUSSION

The presence of the wart virus antigen in human warts has been demonstrated by immunofluorescence techniques (Oroszlan & Rich, 1964; Walter, Walker and Cooper, 1965; Genner, 1971). In all these studies, they have used animal sera to semi-purified wart virus. Genner (1971) looking for antibody to wart virus had success with one human serum. We reported in our previous study (Matthews & Shirodaria, 1973) that human sera can be used successfully in an immunofluorescence test against human warts. In present studies, rabbit serum to highly purified wart virus and positive human sera were used in an indirect immunofluorescence test. The results clearly show that virus antigen detected by rabbit immune serum can also be detected by positive human sera. The warts which had no virus antigen were consistently negative with both rabbit anti-wart virus serum or positive human sera. The examination of a single wart or multiple warts and also the examination of multiple serial sections from a single wart showed that the specific staining was consistent in any one patient and any one lesion and was easily reproducible either with rabbit immune serum or positive human sera. The specific staining properties of immune rabbit serum or positive human sera were both removed by absorption of these sera with purified wart virus. Both antigen-positive and antigen-negative warts were found in all ages and also in warts from different anatomical sites; although the wart lesions from sole, heel and toe had much higher incidence of stainable virus antigen. It was interesting that all regressing wart lesions contained wart virus antigen (Table 2). Barrera-Oro, Smith & Melnick (1962) in their electron microscopic studies showed that only 570% of males and 5000 of females had detectable virus particles in their warts. In most of the warts, antigen was found in the keratinized and granular layers of the skin. But contrary to reports by Walter et al. (1965) and Genner (1971), there was clear evidence in our studies of occasional virus synthesis in the prickle cell layer. All the specific staining in the nuclei of the lower region of the granular layer strongly suggested the presence of virus antigen in the nucleoli. Almeida, Howatson & Williams (1962), in their electron microscopic studies have already shown that the virus is first produced in the nucleolar region. Our findings and those of other workers (Walter et al., 1965; Genner, 1971) that virus antigen is

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337

largely found in the upper keratinized and granular layers of the wart lesion and little or no antigen is detected in the lower proliferative layer raises the question as to whether the cells in the proliferative layer are virus-transformed cells. The failure to detect antigen in the proliferative lower layer of the wart and in wart-negative lesions may be because very little virus-specific antigen is expressed and hence the fluorescent antibody method is not sensitive, or it may be because very early virus-induced non-structural antigens are synthesized in these cells and the antisera used in the test are not adequate for the detection of these antigens. However, to prove this point, it is necessary to demonstrate virus-induced non-structural proteins, such as virus-specified enzymes, in wart lesions. For example, the presence of arginase, antigenically and physiochemically distinct from the cellular arginase has been reported in the rabbit papilloma (Rogers, 1962, 1971). It is also necessary to determine by in situ hybridization techniques whether there is persisting viral genome in wart virus-negative lesions and also in the lower proliferative layer of the wart lesion. So far as we know, ours is the first survey of its kind, where the presence of the virus antigen and of virus-specific antibodies have been compared in the same individual patients. There was a strong correlation between the presence of the virion antigens in the wart and the presence of virus-specific antibodies in corresponding patients' sera (Tables 3 and 4). Some patients who had virus antigen in their lesions had no virus-specific antibody in their sera. As suggested by Ogilvie (1970), this may be due to a reduced antigenic stimulus because the virus is found in a site remote from the blood stream. Also Furminger's (1970) work on guinea-pigs has shown that the immunoglobulin response to wart virus depends on the quantity of virus injected. The patients with regressing warts had both virus-specific IgM and virus-specific IgG antibody. Although in patients with non-regressing warts, virusspecific IgM antibody was predominant, some patients' sera did also contain virus-specific IgG antibody. The process of rejection begins at a molecular level. Since IgG antibody appears before the clinical signs of rejection are observed, it will be interesting to determine the interval between the appearance of virus-specific IgG antibody and the clinical signs of rejection. All the patients who had no virus antigen in their lesion, had no virus-specific antibody in their sera. Pass, Janis and Marcus (1971) demonstrated the presence of non-viral antigens in wart tissue with rabbit antibody prepared against wart tissue. These antigens were also found in normal skin and keratoacanthomata and squamous cell carcinomata but in small amounts. In our survey, anticellular IgM antibody was the commonest type of antibody present in the antigen-positive and antigen-negative groups of patients. The preliminary experiment showed that a fibrillar type of staining pattern was observed when some of these sera were tested on human embryonic lung fibroblast cultures. The staining of microfilamentous network has been reported in acute infective hepatitis (Farrow, Holborow & Brighton, 1971), in patients with multiple sclerosis (Millar et al., 1971) and in acute mumps and measles infections (Haire, 1972). There probably are several different types of anticellular antibodies present in patients with wart virus infection and some of these may be autoantibodies. Only further work will show whether antibodies specific to wart cells are present in patients' sera. The role of anticellular IgM antibodies in the wart virus infection is not clear from the present studies. It was present in groups of patients who had virus antigen in their lesions and virus-specific antibodies in their sera and also in groups of patients who had no stainable virus antigen in their lesions and virus-specific antibodies in their sera. This suggests that the anticellular IgM antibody response may precede the antiviral response. It may be that antiL

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cellular antibody reacting with the infected cell induces expression of virion antigens in wart cells followed by the appearance of virus-specific IgM and IgG antibodies. But further investigations are certainly required to understand the importance of anticellular antibodies in the wart virus infection. A final point of importance is that our results point to a serological rejection mechanism similar to Arthus response, namely, antigen-antibody precipitates and thrombotic lesions in blood vessels of the papilloma. The role of cell-mediated immunity in wart rejection is not understood, although a recent investigation in which lymphocytes from patients with regressing warts did not respond to purified wart virus suggests that cell-mediated immunity may be relatively unimportant (Samuels, unpublished observations). A more definitive work is required to exclude a cell-mediated form of rejection. ACKNOWLEDG MENTS

We wish to thank Professor K. B. Fraser for his encouragement and advice; Dr J. M. Beare and Dr D. Burrows for permission to work with their patients; Miss E. Hoey for doing the sucrose-density gradients; and Mr T. McLoughlin and Mr R. Wood for preparing photo-

graphic prints. REFERENCES ALMEIDA, J.D., HOWATSON, A.F. & WILLIAMS, M.G. (1962) Electron microscopic study of human warts; sites of virus production and nature of the inclusion bodies. J. invest. Derm. 38, 337. BARRERA-ORO, J.G., SMITH, K.O. & MELNICK, J.L. (1962) Quantitation of papova virus particles in human warts. J. nat. Cancer Inst. 29, 583. CHANTLER, S. & HAIRE, M. (1972) Evaluation of the immunological specificity of fluorescein-labelled antihuman IgM conjugates. Immunology, 23, 7. CUBIE, H.A. (1972) Serological studies in a student population prone to infection with human papilloma virus. J. Hyg. (Camb.), 70, 677. FARROW, L.J., HOLBOROW, E.J. & BRIGHTON, W.D. (1971) Reaction of human smooth muscle antibody with liver cells. Nature (Lond.), 232, 186. FURMINGER, I.G.S. (1970) Immunoglobulin synthesis after immunization with human wart virus. Progr.

immunobiol. Standard, 4, 166. GENNER, J. (1971) Localisation of wart virus antigen in human plantar warts (Verrucae vulgares). Acta derm. venereol. (Stockh.), 51, 361. HAIRE, M. (1972) Fibrillar anti-cellular antibody associated with mumps and measles infection. Clin. exp. Immunol. 12, 335. MATTHEWS, R.S. & SHIRODARIA, P.V. (1973) Study of regressing warts by immunofluorescence. Lancet, i, 689. MILLAR, J.H.D., FRASER, K.B., HAIRE, M., CONNOLLY, J.H., SHIRODARIA, P.V. & HADDEN, D.S.M. (1971) Immunoglobulin M specific for measles and mumps in multiple sclerosis. Brit. med. J. ii, 378. OGILVIE, M.M. (1970) Serological studies with human papova (wart) virus. J. Hyg. (Camb.), 68, 479. OROSZLAN, S. & RICH, M.A. (1964). Human wart virus: in vitro cultivation. Science, 146, 531. PASS, F., JANIS, R. & MARCUS, D.M. (1971). Antigens of human wart tissue. J. invest. Derm. 56, 305. ROGERS, S. (1962) Certain relations between the Shope virus-induced arginase, the virus and the tumour cells. The Molecular Basis of Neoplasia (ed. by R. W. Cumley et al.), M. D. Anderson Symposium, p. 483. University of Texas Press, Austin, Texas. ROGERS, S. (1971) Change in the structure of Shope papilloma virus-induced arginase associated with mutation of the virus. J. exp. Med. 134, 1442. SHIRODARIA, P.V., FRASER, K.B. & STANFORD, F. (1973) Secondary fluorescent staining of virus antigens by rheumatoid factor and fluorescein-conjugated anti-IgM. Ann. rheum. Dis. 32, 53. WALTER, E.L., WALKER, D.L. & COOPER, G.A. (1965) Localisation of specific antigen in human warts. Arch. Path. 79, 419.

An immunofluorescence study of warts.

An investigation of the incidence of wart virus-specific antibody and of virion antigens in patients with single or multiple warts taken from differen...
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